- Miscellaneous 756
- Geoduck Genome Sequencing 96
- Olympia Oyster Genome Sequencing 69
- Olympia oyster reciprocal transplant 67
- PROPS 36
- Computer Servicing 24
- Crassostrea gigas larvae OA (2011) bisulfite sequencing 24
- Samples Submitted 23
- LSU C.virginica Oil Spill MBD BS Sequencing 22
- 2bRAD Library Tests for Sequencing at Genewiz 22
- Genotype-by-sequencing at BGI 22
- Goals 20
- Samples Received 15
- Protein expression profiles during sexual maturation in Geoduck 14
- Lineage-specific DNA methylation patterns in developing oysters 11
- BS-seq Libraries for Sequencing at Genewiz 11
- Sea star RNA-seq 10
- Reagent Prep 8
- MBD Enrichment for Sequencing at ZymoResearch 8
- SRA Submissions 6
- Myostatin Interacting Proteins 3
- Tanner Crab RNAseq 3
- 1
- 1
- Olypia Oyster Genome Sequencing 1
- Data Received 1
Miscellaneous
Transcriptome Assessment - BUSCO Metazoa on C.bairdi MEGAN Transcriptome
I previously created a C.bairdi de novo transcriptome assembly with Trinity from the MEGAN6 taxonomic-specific reads for Arthropoda on 20200330 and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.
Transcriptome Annotation - Hematodinium MEGAN Trinity Assembly Using DIAMOND BLASTx on Mox
As part of annotating the most recent transcriptome assembly from the MEGAN6 Hematodinium taxonomic-specific reads, I need to run DIAMOND BLASTx to use with Trinotate.
Transcriptome Assessment - BUSCO Metazoa on Hematodinium MEGAN Transcriptome
I previously created a C.bairdi de novo transcriptome assembly with Trinity from the MEGAN6 taxonomic-specific reads for Alveolata on 20200331 and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.
Transcriptome Assembly - Hematodinium with MEGAN6 Taxonomy-specific Reads with Trinity on Mox
Ran a de novo assembly using the extracted reads classified under Alveolata from:
Transcriptome Assembly - C.bairdi with MEGAN6 Taxonomy-specific Reads with Trinity on Mox
Ran a de novo assembly using the extracted reads classified under Arthropoda from:
RNAseq Reads Extractions - C.bairdi Taxonomic Reads Extractions with MEGAN6 on swoose
Transcriptome Annotation - C.bairdi Using DIAMOND BLASTx on Mox and MEGAN6 Meganizer on swoose
After receiving/trimming the latest round of C.bairdi RNAseq data on 20200318, need to get the data ready to perform taxonomic selection of sequencing reads. To do this, I first need to run DIAMOND BLASTx, then “meganize” the output files in preparation for loading into MEGAN6, which will allow for taxonomic-specific read separation.
Trimming/FastQC/MultiQC - C.bairdi RNAseq FastQ with fastp on Mox
After receiving our RNAseq data from Genewiz earlier today, needed to run FastQC, trim, check trimmed reads with FastQC.
DNA Isolation and Quantification - C.bairdi Hemocyte Pellets in RNAlater
Isolated DNA from 22 samples (see Qubit spreadsheet in “Results” below for sample IDs) using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.
qPCR - C.bairdi RNA Check for Residual gDNA
Previuosly checked existing crab RNA for residual gDNA on 20200226 and identified samples with yields that were likely too low, as well as samples with residual gDNA. For those samples, was faster/easier to just isolate more RNA and perform the in-column DNase treatment in the ZymoResearch Quick DNA/RNA Microprep Plus Kit; this keeps samples concentrated. So, I isolated more RNA on 20200306 and now need to check for residual gDNA.
Trimming/MultiQC - Methcompare Bisulfite FastQs with fastp on Mox
Steven asked me to trim a set of FastQ files, provided by Hollie Putnam, in preparation for methylation analysis using Bismark. The analysis is part of a coral project comparing DNA methylation profiles of different species, as well as comparing different sample prep protocols. There’s a dedicated GitHub repo here:
RNA Isolation and Quantification - C.bairdi RNA from Hemolymph Pellets in RNAlater
Based on qPCR results testing for residual gDNA from 20200225, a set of 24 samples were identified that required DNase treatment and/or additional RNA. I opted to just isolate more RNA from all samples, since the kit includes a DNase step and avoids diluting the existing RNA using the Turbo DNA-free Kit that we usully use. Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.
Data Wrangling - Create Canonical Olurida_v081 Genes FastA
I finally had some time to tackle this GitHub Issue and create a canonical genes FastA file using the MAKER IDs, instead of the original contig IDs from our Olympia oyster genome assembly - https://owl.fish.washington.edu/halfshell/genomic-databank/Olurida_v081.fa (FastA; 1.1GB).
qPCR - C.bairdi RNA Check for Residual gDNA
After deciding on a primer set to use for gDNA detection on 20200225, went ahead and ran a qPCR on most of the RNA samples described in Grace’s Google Sheet. Some samples were not run, as they had not yet been located at the time I began the qPCR.
qPCR - C.bairdi Primer Tests on gDNA
We received the primers I ordered on 20200220 and now need to test them to see if they detect gDNA. If yes, then they’re good candidates to assess the presence of residual gDNA in our RNA samples before we proceed with reverse transcription.
Primer Design - C.bairdi Primers for Checking RNA for Residual gDNA
Getting ready to run some qPCRs and first we need to confirm that our RNA is actually DNA-free. Before we can do that, we need some primers to use, so I decided to semi-arbitrarily select three different gene targets from our MEGAN6 taxonomic-specific Trinity assembly from 20200122.
DNA Isolation & Quantification - C.bairdi RNA from Samples 6212_132_9 6212_334_12 6212_485_26
Isolated DNA from three samples (see Qubit spreadsheet in “Results” below for sample IDs) using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.
RNA Isolation & Quantification - C.bairdi RNA from Samples 6212_132_9 6212_334_12 6212_485_26
We are supposed to get RNA sent out for sequencing today, but it turns out that a few of the designated samples have insufficient RNA in them. So, I’m going to attempt to isolate enough RNA from the following samples in order to have enough RNA to send to Genewiz today:
RNA Isolation & Quantification - C.bairdi RNA from Sample 6129_403_26
Since I was isolating gDNA from C.bairdi 6129_403_26 hemolymph, I figured I might as well co-isolate RNA since I was using the Quick DNA/RNA Microprep Plus Kit (ZymoResearch).
DNA Isolation & Quantification - Additional C.bairdi gDNA from Sample 6129_403_26
Earlier today I isolated gDNA from C.bairi 6129_403_26 hemolymph pellets and recovered decently intact gDNA that could be used for sequencing. However, I still need more gDNA, so will isolate that (and co-isolate RNA, since I’m going through the procedure anyway) using the rest of the sample using the Quick DNA/RNA Microprep Plus Kit (ZymoResearch).
DNA Isolation, Quantification, and Gel - C.bairdi gDNA Sample 6129_403_26
In order to do some genome sequencing on C.bairid and Hematodinium, we need hihg molecular weight gDNA. I attempted this twice before, using two different methods (Quick DNA/RNA Microprep Kit (ZymoResearch) on 20200122 and the E.Z.N.A Mollusc DNA Kit (Omega) on 20200108) using ~10yr old ethanol-preserved tissue provided by Pam Jensen. Both methods yielded highly degrade gDNA. So, I’m now attempting to get higher quality gDNA from the RNAlater-preserved hemolymph pellets from this experiment.
Transcriptome Assessment - BUSCO Metazoa on Hematodinium MEGAN Transcriptome
I previously created a Hematodinium de novo transcriptome assembly with Trinity from the MEGAN6 taxonomic-specific reads for Alveolata on 20200122 and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.
Transcriptome Assessment - BUSCO Metazoa on C.bairdi MEGAN Transcriptome
I previously created a C.bairdi de novo transcriptome assembly with Trinity from the MEGAN6 taxonomic-specific reads for Arthropoda on 20200122 and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.
Gene Expression - Hematodinium MEGAN6 with Trinity and EdgeR
After completing annotation of the Hematodinium MEGAN6 taxonomic-specific Trinity assembly using Trinotate on 20200126, I performed differential gene expression analysis and gene ontology (GO) term enrichment analysis using Trinity’s scripts to run EdgeR and GOseq, respectively. The comparison listed below is the only comparison possible, as there were no reads present in the uninfected Hematodinium extractions.
Gene Expression - C.bairdi MEGAN6 with Trinity and EdgeR
After completing annotation of the C.bairdi MEGAN6 taxonomic-specific Trinity assembly using Trinotate on 20200126, I performed differential gene expression analysis and gene ontology (GO) term enrichment analysis using Trinity’s scripts to run EdgeR and GOseq, respectively, across all of the various treatment comparisons. The comparison are listed below and link to each individual SBATCH script (GitHub) used to run these on Mox.
Data Wrangling - Arthropoda and Alveolata Day and Treatment Taxonomic RNAseq FastQ Extractions
After using MEGAN6 to extract Arthropoda and Alveolata reads from our RNAseq data on 20200114, I had then extracted taxonomic-specific reads and aggregated each into basic Read 1 and Read 2 FastQs to simplify transcriptome assembly for C.bairdi and for Hematodinium. That was fine and all, but wasn’t fully thought through.
DNA Isolation and Quantification - C.bairdi Hemocyte Pellets in RNAlater
Isolated DNA from 56 samples (see Qubit spreadsheet in “Results” below for sample IDs) using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.
Transcriptome Annotation - Trinotate Hematodinium MEGAN6 Taxonomic-specific Trinity Assembly on Mox
After performing de novo assembly on our Hematodinium MEGAN6 taxonomic-specific RNAseq data on 20200122 and performing BLASTx annotation on 20200123, I continued the annotation process by running Trinotate.
Transcriptome Annotation - Trinotate C.bairdi MEGAN6 Taxonomic-specific Trinity Assembly on Mox
After performing de novo assembly on our Tanner crab MEGAN6 taxonomic-specific RNAseq data on 20200122 and performing BLASTx annotation on 20200123, I continued the annotation process by running Trinotate.
RNA Isolation and Quantification - C.bairdi Hemocyte Pellets in RNAlater
Isolated RNA from the following hemolymph pellet samples:
RNA Isolation and Quantification - C.bairdi Hemocyte Pellets in RNAlater
Isolated RNA from the following hemolymph pellet samples:
Transdecoder - Hematodinium MEGAN6 Taxonomic-Specific Reads Assembly from 20200122
Ran Trinity to de novo assembly on the the C.bairdi MEGAN6 taxonomic-specific RNAseq data on 201200122 and now will begin annotating the transcriptome using TransDecoder on Mox.
Transdecoder - C.bairdi MEGAN6 Taxonomic-Specific Reads Assembly from 20200122
Ran Trinity to de novo assembly on the the Hematodinium MEGAN6 taxonomic-specific RNAseq data on 201200122 and now will begin annotating the transcriptome using TransDecoder on Mox.
Transcriptome Annotation - Hematodinium MEGAN Trinity Assembly Using DIAMOND BLASTx on Mox
As part of annotating the transcriptome assembly from the MEGAN6 Hematodinium taxonomic-specific reads, I need to run DIAMOND BLASTx to use with Trinotate.
Transcriptome Annotation - C.bairdi MEGAN Trinity Assembly Using DIAMOND BLASTx on Mox
As part of annotating the transcriptome assembly from the MEGAN6 C.bairdi taxonomic-specific reads, I need to run DIAMOND BLASTx to use with Trinotate.
RNA Isolation and Quantification - C.bairdi Hemocyte Pellets in RNAlater Troubleshooting
After the failure to obtain RNA from any C.bairdi hemocytes pellets (out of 24 samples processed) on 20200117, I decided to isolate RNA from just a subset of that group to determine if I screwed something up last time or something. Also, I am testing two different preparations of the kit-supplied DNase I: one Kaitlyn prepped and a fresh preparation that I made. Admittedly, I’m not doing the “proper” testing by trying the different DNase preps on the same exact sample, but it’ll do. I just want to see if I get some RNA from these samples this time…
DNA Quality Assessment - Agarose Gel for C.bairdi 20102558-2729 gDNA from 20200122
Earlier today, I isolated gDNA from C.bairdi 20102558-2729 ethanol-preserved muscle tissue using the Quick DNA/RNA MicroPrep Plus Kit (ZymoResearch) and prepared the tissue in three different ways to see how they would compare:
Data Wrangling - Arthropoda and Alveolata Taxonomic RNAseq FastQ Extractions
After using MEGAN6 to extract Arthropoda and Alveolata reads from our RNAseq data on 20200114, I realized that the FastA headers were incomplete and did not distinguish between paired reads. Here’s an example:
DNA Isolation - C.bairdi 20102558-2729 EtOH-preserved Tissue via Three Variations Using Quick DNA-RNA MicroPrep Kit
Previously, I isolated gDNA from a C.bairdi EtOH-preserved muscle sample (20102558-2729) on 20200108 using the E.Z.N.A. Mollusc DNA Kit (Omega). Although the yields were excellent, the DNA looked completely degraded on a gel and running that DNA on a minION flowcell yielded relatively short reads (which wasn’t terribly surprising).
Transcriptome Assembly - Hematodinium with MEGAN6 Taxonomy-specific Reads with Trinity on Mox
Ran a de novo assembly using the extracted reads classified under Alveolata from 20200122 The assembly was performed with Trinity on Mox.
Transcriptome Assembly - C.bairdi with MEGAN6 Taxonomy-specific Reads with Trinity on Mox
Ran a de novo assembly using the extracted reads classified under Arthropoda from 20200122 The assembly was performed with Trinity on Mox.
DNA Isolation and Quantification - C.bairdi Hemolymph Pellets in RNAlater
Isolated DNA from the following 23 samples:
RNA Isolation and Quantification - C.bairdi Hemolymph Pellets in RNAlater
TL;DR - Recovered absolutely no RNA from any sample! However, I did recover DNA from each sample.
RNAseq Reads Extractions - C.bairdi Taxonomic Reads Extractions with MEGAN6 on swoose
I previously ran BLASTx and “meganized” the output DAA files on 20200103 and now need to use MEGAN6 to bin the results into the proper taxonomies. This is accomplished using the MEGAN6 graphical user interface (GUI). This is how the process goes:
Lab Maintenance - Cluster UPS Battery Replacement
Replaced the batteries on one of the APC uninterruptable power supplies (UPS) on our local server cabinet.
DNA Quality Assessment - Agarose Gel and NanoDrop on C.bairdi gDNA
I isolated C.bairdi gDNA yesterday (20200108) and now want to get an idea if it’s any good (i.e. no contaminants, high molecule weight).
DNA Isolation and Quantification - C.bairdi gDNA from EtOH Preserved Tissue
I isolated gDNA from ethanol-preserved C.bairdi muscle tissue from sample 20102558-2729 (SPNO-ReferenceNO). This sample was chosen as it had 0 in the SMEAR_result and BCS_PCR_results columns, indicating it should be free of Hematodinium. See the sample spreadsheet linked below for more info.
Transcriptome Annotation - C.bairdi Using DIAMOND BLASTx on Mox and MEGAN6 Meganizer
Although I previously annotated our C.bairdi transcriptome from 20191218, I realized that the assembly and annotations were combine infected/uninfected samples, possibly making separating crab/Hematodinium sequences a bit more difficult.
Transcriptome Annotation - C.bairdi Trinity Assembly Trinotate on Mox
After performing de novo assembly on our Tanner crab RNAseq data on 20191218 and performing BLASTx annotation on 20191224, I continued the annotation process by running Trinotate.
Transcriptome Annotation - C.bairdi Trinity Assembly BLASTx on Mox
In preparation for complete transcriptome annotation of the C.bairdi de novo assembly fro 20191218, I needed to run BLASTx. The assembly was BLASTed against the SwissProt database that comes with Trinotate. Initial BLAST output format selected was format 11 (i.e. ASN format), as this allows for simple conversion between different formats later on, if desired.
Transdecoder - C.bairdi De Novo Transcriptome from 20191218 on Mox
Ran Trinity to de novo assemble the C.bairdi RNAseq data we had on 20191218 and now will begin annotating the transcriptome using TransDecoder on Mox.
Transcriptome Assembly - C.bairdi Trimmed RNAseq Using Trinity on Mox
Earlier today, I trimmed our existing C.bairdi RNAseq data, as part of producing generating a transcriptome (per this GitHub issue). After trimming, I performed a de novo assembly using Trinity (v2.9.0) with the stranded library option (--SS_lib_type RF) on Mox.
Trimming/FastQC/MultiQC - C.bairdi RNAseq FastQ with fastp on Mox
Grace/Steven asked me to generate a de novo transcriptome assembly of our current C.bairdi RNAseq data in this GitHub issue. As part of that, I needed to quality trim the data first. Although I could automate this as part of the transcriptome assembly (Trinity has Trimmomatic built-in), I would be unable to view the post-trimming results until after the assembly was completed. So, I opted to do the trimming step separately, to evaluate the data prior to assembly.
Data Wrangling - Olurida_v081 UTR GFFs and Intergenic, Intron BED files
After a meeting last week, we realized we needed to update the paper-oly-mbdbs-gen GitHub repo with the most current versions of feature files we had.
PCR - Crassostrea gigas and sikamea Mantle gDNA from Marinelli Shellfish Company
I ran this PCR a couple of times before and, embarrassingly, I had ordered/used the wrong primers.
qPCR - Geoduck hemolymph and hemocyte cDNA with vitellogenin primers
Previously isolated RNA on 20191125 and made cDNA on 20191126 from some geoduck hemolymph and hemocyte samples that Shelly asked me to run qPCRs on.
Reverse Transcription - P.generosa DNased Hemolypmh and Hemocyte RNA from 20191125
Performed reverse transcription on the DNased hemolymph and hemocyte RNA from yesterday.
RNA Isolation and Quantification - Geoduck hemolymph and hemocyte samples
Shelly asked me to isolate RNA and run some qPCRs on the following samples:
PCR - Crassostrea gigas and sikamea Mantle gDNA from Marinellie Shellfish Company - No Multiplex
UPDATE 20191125
PCR - Crassostrea gigas and sikamea Mantle gDNA from Marinelli Shellfish Company
UPDATE 20191125
PCR - Crassostrea gigas and sikamea Mantle gDNA from Marinelli Shellfish Company
UPDATE 20191125
DNA Isolation and Quantification - Crassostrea gigas and Crassostrea sikamea Mantle Tissue from Marinelli Shellfish Company
Isolated DNA from the C.gigas and C.sikamea samples we received from Marinelli Shellfish Company on 20191030 using DNAzol.
Samples Received - Marinelli Shellfish Company C.gigas and C.sikamea Oysters
Steven was recently contacted by Marinelli Shellfish Company to see if we could help them determine if some oysters they had were Crassostrea gigas (Pacific oyster) or Crassostrea sikamea (Kumamoto). Steven knows of a paper with primer sequences to use with qPCR for this specific determination.
FastQC-MultiQC - C.bairdi RNAseq Day 12 26 Infected Uninfected
After receiving the rest of the crab data and concatenating it all together, I ran FastQC and MultiQC on the FastQ files.
Data Received - C.bairdi RNAseq Day9-12-26 Infected-Uninfected
Previously, we “received” this data, but it turns out it was incomplete (see 20191003).
Lab Maintenance - Cluster UPS Battery Replacement
Replaced the batteries on one of the APC uninterruptable power supplies (UPS) on our local server cabinet.
Metagenomics Annotation - P.generosa Water Samples with MEGAN6
After running DIAMOND BLASTx and MEGANIZER on these samples on 20190925 to assess taxonomy info, I began the analyses/visualization of this data with MEGAN6.
Data Received - C.bairdi RNAseq Day9-12-26 Infected-Uninfected
UPDATE (20191024): This post details receipt of incomplete data. Additional sequencing was performed and that additional data was received 20191024. This notebook entry on 20191024 contains details on FastQ concatenation of the data below and the data received on 20191024.
Metagenomics Annotation - P.generosa Water Samples Using DIAMOND BLASTx on Mox
Trimming/FastQC/MultiQC - P.generosa EPI FastQs with FASTP on Mox
Steven noticed that the M-Bias plots generated by Bismark from these files was a little wonky and asked that I try trimming them a bit more. The files were originally quality/adaptor trimmed with TrimGalore! on 20180516.
Genome Comparison - Pgenerosa_v074 vs Pgenerosa_v070 with MUMmer Promer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer 3.23 (specifically, promer for protein level comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs S.glomerata NCBI with MUMmer Promer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer 3.23 (specifically, promer for protein level comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs M.yessoensis NCBI with MUMmer Promer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer 3.23 (specifically, promer for protein level comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs H.sapiens NCBI with MUMmer Promer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer 3.23 (specifically, promer for protein level comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs C.virginica NCBI with MUMmer Promer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer 3.23 (specifically, promer for protein level comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs C.gigas NCBI with MUMmer Promer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer 3.23 (specifically, promer for protein level comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs Pgenerosa_v070 with MUMmer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer (v4) (specifically, nucmer for nucleotide comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs Pgenerosa_v074 with MUMmer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer (v4) (specifically, nucmer for nucleotide comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs S.glomerata NCBI with MUMmer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer (v4) (specifically, nucmer for nucleotide comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs M.yessoensis NCBI with MUMmer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer (v4) (specifically, nucmer for nucleotide comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs H.sapiens NCBI with MUMmer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer (v4) (specifically, nucmer for nucleotide comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs C.gigas NCBI with MUMmer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer (v4) (specifically, nucmer for nucleotide comparisons). This software is specifically designed to do this type of comparison.
Genome Comparison - Pgenerosa_v074 vs C.virginica NCBI with MUMmer on Mox
In continuing to further improve our geoduck genome annotation, I’m attempting to figure out why Scaffold 1 of our assembly doesn’t have any annotations. As part of that I’ve decided to perform a series of genome comparisons and see how they match up, with an emphasis on Scaffold 1, using MUMmer (v4) (specifically, nucmer for nucleotide comparisons). This software is specifically designed to do this type of comparison.
Data Wrangling - FastA Splitting With faSplit
Steven posted an issue on GitHub regarding splitting a FastA file into multiple sequences. Specifically, he wanted a single, large FastA sequence (~89Mbp) split into smaller FastAs for BLASTing.
Data Summary - P.generosa Transcriptome Assemblies Stats
In our continuing quest to wrangle the geoduck transcriptome assemblies we have, I was tasked with compiling assembly stats for our various assemblies. The table below provides an overview of some stats for each of our assemblies. Links within the table go to the the notebook entries for the various methods from which the data was gathered. In general:
Transcriptome Compression - P.generosa Transcriptome Assemblies Using CD-Hit-est on Mox
In continued attempts to get a grasp on the geoduck transcriptome size, I decided to “compress” our various assemblies by clustering similar transcripts in each assembly in to a single “representative” transcript, using CD-Hit-est. Settings use to run it were taken from the Trinity FAQ regarding “too many transcripts”.
Transcriptome Annotation - Geoduck Larvae Day5 EPI99 with Transdecoder on Mox
Used Transdecoder to identify open reading frames (ORFs) for use in annotating Pgenerosa_v074 genome assembly. Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Super Low OA EPI116 with Transdecoder on Mox
Used Transdecoder to identify open reading frames (ORFs) for use in annotating Pgenerosa_v074 genome assembly. Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Juvenile Super Low OA EPI115 with Transdecoder on Mox
Used Transdecoder to identify open reading frames (ORFs) for use in annotating Pgenerosa_v074 genome assembly. Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Juvenile Ambient OA EPI124 with Transdecoder on Mox
Used Transdecoder to identify open reading frames (ORFs) for use in annotating Pgenerosa_v074 genome assembly. Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Juvenile Ambient OA EPI123 with Transdecoder on Mox
Used Transdecoder to identify open reading frames (ORFs) for use in annotating Pgenerosa_v074 genome assembly. Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Gonad with Transdecoder on Mox
Used Transdecoder to identify open reading frames (ORFs) for use in annotating Pgenerosa_v074 genome assembly. Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Ctenidia with Transdecoder on Mox
Used Transdecoder to identify open reading frames (ORFs) for use in annotating Pgenerosa_v074 genome assembly. Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
RepeatModeler - Pgenerosa_v074 for MAKER Annotation on Emu
Yesterday (20190625) I generated a subset of the first 18 FastA sequences from the Pgenerosa_v070.fa file. This subset has been designated as Pgenerosa_v074 by Steven. It’s available on our Genomic Resources wiki:
RepeatMasker - Pgenerosa_v074 for Transposable Element ID on Roadrunner
Yesterday (20190625) I generated a subset of the first 18 FastA sequences from the Pgenerosa_v070.fa file. This subset has been designated as Pgenerosa_v074 by Steven. It’s available on our Genomic Resources wiki:
Data Received - C.virginica Mantle MBD-BSseq from ZymoResearch
We received the BSseq data from ZymoResearch today. Samples were submitted on 20190326. The samples constituted 24 Crassostrea virginica mantle samples which were prepared using the MethylMiner Kit (Invitrogen) on 20190319.
Data Wrangling - FastA Subsetting of Pgenerosa_v070.fa Using samtools faidx
Steven asked to subset the Pgenerosa_v070.fa (2.1GB) in this GitHub Issue #705. In that issue, it was determined that a significant portion of the sequencing data that was assembled by Phase Genomics clustered in “scaffolds” 1 - 18. As such, Steven asked to subset just those 18 scaffolds.
Metagenomics - Taxonomic Diversity and Sequencing Coverage with MEGAHIT BLASTx and Krona Plots
After a meeting on this project around the middle of May, we decided to try various approaches to assessing the metagenome. One aspect was to add coverage sequencing coverage information to our BLASTx taxonomy visualizations. I used the MEGAHIT coverage info from 20190327 and the subsequent BLASTx data from 20190516.
Sample Submission - Tanner Crab Infected vs Uninfected RNAseq
After reviewing our options for sample pooling, we decided to do a comparison of Infected vs. Uninfected crabs.
Metagenomics - BLASTx of Individual Water Sample MEGAHIT Assemblies on Mox
After a meeting on this project yesterda, we decided to try a few things to continue with various approaches to assessing the metagenome. One of the approaches is to run BLASTx on the individual water sample MEGAHIT assemblies from 20190327 and obtain taxonomy info for them, so that’s what I did here.
Library Decisions - C.bairdi RNAs for Library Pools
FastQC-MultiQC - Additional C.gigas WGBS Sequencing Data from Genewiz Received 20190501
Earlier today, we received the additional G.gigas sequencing data from Genewiz. Wanted to run through FastQC again and get an updated report for each data set. Admittedly, it probably won’t look much different from the initial FastQC run on 20190415, due to the fact that the additional sequencing was simply appended to the previous data. Since FastQC examines a subset of the data in each file, I’d fully expect the FastQC report to look the same. However, we’ll have a greater number of sequences in each file. This should, in turn, increase the number of reads retained after quality trimming.
Data Received - Additional C.gigas Whole Genome Bisulfite Sequencing Data from Genewiz
The FastQC analysis of the intitial data we received from Genewiz (on 20190408)showed consistent failures in the “Per Tile Sequence Quality” for all of Roberto’s Crassostrea gigas sequencing. After discussing with Genewiz, they offered to generate an additional 25% reads for each of those libraries.
RNA Isolation and Quantification - C.bairdi Hemolymph Pellet in RNAlater
After the success we had isolating RNA using the Quick-DNA/RNA Microprep Plus Kit (ZymoResearch), Steven had me isolate RNA from a list of ~117 samples. Of that list, I was able to find 66 crab hemolymph pelleted RNAlater samples. The “missing” samples were most likely previosly used by Grace during our various attempts to get some usable RNA out these.
Data Analysis - C.virginica MBD Sequencing Coverage
A while ago, Steven tasked me with assessing some questions related to the sequencing coverage we get doing MBD-BSseq in this GitHub issue. At the heart of it all was really to try to get an idea of how much usable data we actually get when we do an MBD-BSseq project. Yaamini happened to have done an MBD-BSseq project relatively recently, and it’s one she’s actively working on analyzing, so we went with that data set.
Metagenomics Annotation - P.generosa Water Samples Using BLASTn on Mox and KronaTools Visualization to Compare pH Treatments
Nearing the end of this quick metagenomics comparison of taxonomic differences between the two pH treatments (pH=7.1 and pH=8.2). Previously ran:
Metagenomics Gene Prediction - P.generosa Water Samples Using MetaGeneMark on Mox to Compare pH Treatments
Continuing with a relatively quick comparison of pH treatments (pH=7.1 vs. pH=8.2), I wanted to run gene prediction on the MEGAHIT assemblies I made yesterday. I ran MetaGeneMark on the two pH-specific assemblies on Mox. This should be a very fast process (I’m talking, like a couple of minutes fast), so it enhances the annotation with very little effort and time.
FastQC - WGBS Sequencing Data from Genewiz Received 20190408
We received whole genome bisulfite sequencing (WGBS) data from Genewiz last week on 20190408, so ran FastQC on the files on my computer (swoose). FastQC results will be added to Nightingales Google Sheet.
Metagenome Assemblies - P.generosa Water Samples Trimmed HiSeqX Data Using Megahit on Mox to Compare pH Treatments
A report involving our work on the geoduck water metagenomics is due later this week and our in-depth analysis for this project using Anvi’o is likely to require at least another week to complete. Even though we have a broad overview of the metagenomic taxa present in these water samples, we don’t have data in a format for comparing across samples/treatments. So, I initiated our simplified pipeline (MEGAHIT > MetaGeneMark > BLASTn > KronaTools) for examining our metagenomic data of the two treatments:
RNA Isolation and Quantification - Crab Hemolypmh Using Quick-DNA-RNA Microprep Plus Kit
In the continuing struggle to isolate RNA from the Chionoecetes bairdi hemolymph preserved in RNAlater, Pam managed to find the Quick-DNA-RNA Microprep Plus Kit (ZymoResearch) as a potential option. We received a free sample of the kit and so I gave it a shot. Grace pulled 10 samples she’d previously used to isolate RNA (and was unable to get anything out of virtually all of them using the RNeasy Plus Micro Kit (Qiagen)) for me to try out this new kit:
Transcriptome Assembly - Geoduck Tissue-specific Assembly Larvae Day5 EPI99 with HiSeq and NovaSeq Data on Mox
I previously assembled and annotated P.generosa larval Day 5 transcriptome (20190318 - mislabeled as Juvenile Day 5 in my previous notebook entries) using just our HiSeq data from our Illumina collaboration. This was a an oversight, as I didn’t realize that we also had NovaSeq RNAseq data. So, I’ve initiated another de novo assembly using Trinity incorporating both sets of data.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Gonad HiSeq and NovaSeq Data on Mox
I previously assembled and annotated P.generosa gonad transcriptome (20190318) using just our HiSeq data from our Illumina collaboration. This was a an oversight, as I didn’t realize that we also had NovaSeq RNAseq data. So, I’ve initiated another de novo assembly using Trinity incorporating both sets of data.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Juvenile Ambient OA EPI124 with HiSeq and NovaSeq Data on Mox
Ran a de novo assembly on our HiSeq and NovaSeq data from Hollie’s juvenile EPI 124 sample “ambient OA”. This was done for Christian to use in some long, non-coding RNA (lncRNA) analysis.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Juvenile Ambient OA EPI123 with HiSeq Data on Mox
Ran a de novo assembly on our HiSeq data from Hollie’s juvenile EPI 123 sample “ambient OA”. This was done for Christian to use in some long, non-coding RNA (lncRNA) analysis.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Juvenile Super Low OA EPI116 with HiSeq and NovaSeq Data on Mox
Ran a de novo assembly on our HiSeq and NovaSeq data from Hollie’s juvenile EPI 116 sample “super low OA”. This was done for Christian to use in some long, non-coding RNA (lncRNA) analysis.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Juvenile Super Low OA EPI115 with HiSeq Data on Mox
Ran a de novo assembly on our HiSeq data from Hollie’s juvenile EPI 115 sample “super low OA”. This was done for Christian to use in some long, non-coding RNA (lncRNA) analysis.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Ctenidia with HiSeq and NovaSeq Data on Mox
I previously assembled and annotated P.generosa ctenidia transcriptome (20190318) using just our HiSeq data from our Illumina collaboration. This was a an oversight, as I didn’t realize that we also had NovaSeq RNAseq data. So, I’ve initiated another de novo assembly using Trinity incorporating both sets of data.
Data Management - Whole Genome Bisulfite Sequencing Data from Genewiz Received
Received the WGBS data from Genewiz that were sent to Genewiz for whole genome bisulfite sequencing on 20190207. These were from:
Metagenomics - P.generosa Water Sample Assemlby Comparisons with Quast
Continuing work on the metagenomics project, Emma shared her “co-assembly”, so I figured it would be quick and easy to compare hers with mine and get a feel for how different/similar they might be. I did a similar comparison last week where I compared each of our individual water sample assemblies. Those results showed my assemblies generated:
Metagenomics - Geoduck Water Sample Assembly Comparisons with MetaQuast
As part of addressing this GitHub Issue on assessing taxonomic diversity in our metagenomics samples, I decided to compare the individual sample assemblies I made on 20190327 using Megahit and the assemblies that Emma made. Emma utilized NGless on her cluster in Genome Sciences with the following scripts:
Metagenomics - Taxonomic Diversity Comparisons from Geoduck Water with Anvio on Mox
I decided to give Anvi’o a shot for this metagenomics analysis, as it seems ridiculously thorough and easy to use, with a nice-looking, interactive plotting interface. Additionally, they have a TON of clearly written tutorials on how to use their software and explanations of what’s happening when you use it! Really, couldn’t ask for much more. So, here’s how it went…
Metagenome Assemblies - P.generosa Water Samples Trimmed HiSeqX Data Using Megahit on Mox
I previously created a single metagenome assembly using all of the sequencing data from this project.
Transposable Element Mapping - Crassostrea gigas Genome v9 Using RepeatMasker 4.07 on Roadrunner
Per this GitHub issue, I’m IDing transposable elements (TEs) in the Crassostrea gigas genome. Even though the C.gigas genome should be fully annotated, Steven wants a comparable set of analyses to compare to the Crassostrea virginica TE mapping we previously performed.
Sample Submission - Lotterhos C.virginica Mantle MBD DNA to ZymoResearch for BSseq
Crassostrea virginica samples that were subjected to MBD enrichment (completed 20190319) were shipped, on dry ice, to ZymoResearch today for BSseq. They will perform bisulfite conversion, library prep, and sequencing. Sequencing output is to be ~50M paired-end reads (at least 100bp or 151bp) per library.
Data Management - SRA Submission Panopea generosa Day 5 Larvae RNAseq
In preparation for some manuscripts, Steven asked that I get some geoduck RNAseq data upload to the NCBI sequencing read archive (SRA) in this GitHub Issue. Specifically, he needed the data corresponding to day 5 (pos-fertilization) larve RNAseq data that was prepped/run by Illumina on the NovaSeq. Original sample submission notebook entry is here.
Metagenomics - Taxonomic Diversity from Geoduck Water with BLASTn and Krona Plots
Continuing on getting the metagenomics sequencing project written up as a manuscript, Steven asked me to provide an overview of the taxonomic makeup of our metagenome assembly in this GitHub Issue. Earlier today, I ran analysis using BLASTp data. I initiated additional analysis using the MetaGeneMark nucleotide data to run BLASTn on Mox.
Metagenomics - Taxonomic Diversity from Geoduck Water with BLASTp and Krona plots
We’re working on getting the metagenomics sequencing project written up as a manuscript and Steven asked me to provide an overview of the taxonomic makeup of our metagenome assembly in this GitHub Issue.
MBD Enrichment - DNA Quantification of C.virginica MBD Samples from 20190312
Finished MBD enrichment on 20190312 using the MethylMiner kit. Since we were out of Qubit assay tubes, I could not quantify these samples when I initially completed the ethanol precipitation. Tubes are in, so I went forward with quantification using the Roberts’ Lab Qubit 3.0 and the 1x High Sensitivity dsDNA assay.
Transcriptome Annotation - Geoduck Juvenile Day 5 with Trinotate on Mox
Transcriptome Annotation - Geoduck Heart with Trinotate on Mox
Transcriptome Annotation - Geoduck Gonad with Trinotate on Mox
Transcriptome Annotation - Geoduck Ctenidia with Trinotate on Mox
Transcriptome Annotation - Geoduck Heart with BLASTx on Mox
I’ll be annotating the transcriptome assembly (from 20190215) using Trinotate and part of that process is having BLASTx output for the Trinity assembly, so have run BLASTx on Mox.
Transcriptome Annotation - Geoduck Gonad with BLASTx on Mox
I’ll be annotating the transcriptome assembly (from 20190215) using Trinotate and part of that process is having BLASTx output for the Trinity assembly, so have run BLASTx on Mox.
Transcriptome Annotation - Geoduck Ctenidia with BLASTx on Mox
I’ll be annotating the transcriptome assembly (from 20190215) using Trinotate and part of that process is having BLASTx output for the Trinity assembly, so have run BLASTx on Mox.
Transcriptome Annotation - Geoduck Juvenile Day 5 with BLASTx on Mox
I’ll be annotating the transcriptome assembly (from 20190215) using Trinotate and part of that process is having BLASTx output for the Trinity assembly, so have run BLASTx on Mox.
Transcriptome Annotation - Geoduck Juvenile Day 5 with Transdecoder on Mox
Continuing tissue-specific RNAseq. Using Transdecoder to identify open reading frames (ORFs). Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Heart with Transdecoder on Mox
Continuing tissue-specific RNAseq. Using Transdecoder to identify open reading frames (ORFs). Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Gonad with Transdecoder on Mox
Continuing tissue-specific RNAseq. Using Transdecoder to identify open reading frames (ORFs). Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Transcriptome Annotation - Geoduck Ctenidia with Transdecoder on Mox
Continuing tissue-specific RNAseq. Using Transdecoder to identify open reading frames (ORFs). Relies on BLASTp, Pfam, and HMM scanning to ID ORFs.
Methylation Analysis - C.virginica Gonad MBD with Varying Read Subsets with Bismark on Mox
Steven asked for the following analysis in this GitHub Issue using Yaamini’s C.virginica MBD samples:
Data Management - Create C.virginica Bisulfite Genome with Bismark on Mox
In preparation for some a bisulfite analysis requested by Steven, I needed to make bisulfite genome with, and for, Bismark to use and wanted to use the same one Yaamini has been working with. Downloaded the NCBI version of the C.virginica genome.
MBD Enrichment - Ethanol Precipitation of C.virginica MBD samples
After completing two rounds of MBD enrichment (first 12 samples on 20190307 and the second 12 samples on 20190311), I performed ethanol precipitation, per the MethylMiner instructions with the following modifications:
MBD Enrichment - C.virginica Sheared Mantle DNA from 20190306
Last week I performed MBD enrichment on 12 of the 24 Lotterhos C.viriginica mantle DNA on samples I had sheared on 20190306. I proceeded with MBD enrichment using the MethylMiner Kit (Invitrogen) on the 12 remaining samples of the 24. I followed the manufacturer’s protocol for input DNA >1ug - 10ug with the following modifications:
MBD Enrichment - C.virginica Sheared Mantle DNA from 20190306
Yesterday, I sheared the Lotterhos C.virginica gDNA in preparation for MBD enrichment. Today, I proceeded with MBD enrichment using the MethylMiner Kit (Invitrogen) on 12 of the 24 samples. I followed the manufacturer’s protocol for input DNA >1ug - 10ug with the following modifications:
DNA Shearing & Bioanalyzer - Lotterhos C.virginica Mantle gDNA from 2018114
Crassostrea virginica mantle gDNA that had previously been isolated on 20181114 was evaluated for integrity via agaroase gel yesterday (20190305). Everything looked pretty good, so proceeded with shearing in preparation for MBD enrichment.
Agarose Gel - Lotterhos C.virginica Mantle DNA from 20181114
Checked DNA integrity of the Crassostrea virginica mantle gDNA I isolated on 20181114 in preparation for MBD enrichment. Detailed sample info and sample processing (including calculations for MBD enrichment using the MethylMiner Kit) are here (Google Sheet):
Data Management - Data Migration and Drive Expansion on Gannet
A little while ago, we installed some additional hard drives in Gannet (Synology RS3618XS) with the intention of expanding the total storage space. However, the original set of HDDs were set up as RAID10. As it turns out, RAID10 configurations cannot be expanded! So, the new set of HDDs were configured as a separate volume (Volume 2) in a RAID6 configuration. After backing up the /volume1/web directory (via rsync) to our UW Google Drive, I begane the data migration.
Genome Assessment - BUSCO Metazoa on P.generosa v071 on Mox
Ran BUSCO on our completed annotation of the P.generosa v071 genome (GFF) (subset of sequences >10kbp). See this notebook entry for genome annotation info. This provides a nice metric on how “complete” a genome assembly (or transcriptome) is. Additionally, BUSCO is tied in with Augustus for gene prediction and generates ab initio gene models. With that said, since I just want to evaluate the completeness of this particular genome assembly, I’ll be using the annotated genome generated through two rounds of SNAP gene prediction. Otherwise, I’d use the initial MAKER annotations to generate an Augustus gene model that could be used in conjuction with the SNAP models (I’ll likely do this at a later date).
Data Wrangling - CpG OE Calculations on C.virginica Genes
Steven tasked me with processing ~90 FastA files containing gene sequences from C.virginica in this GitHub Issue. He needed to determine the Observed/Expected (O/E) ratio of CpGs in each FastA. He provided this example code and this link to all the files. Additionally, today, he tasked Kaitlyn with merging all of the output CpG O/E values for each sample in to a single file, but I decided to tackle it anyway.
Methylation Analysis - P.generosa Bismark on Mox
This is a quick and dirty (i.e. no adaptor/quality trimming) assessment of all of our Panopea generosa bisulfite sequencing efforts to date in order to get a rough idea of methylation mapping, per this GitHub issue. Ran Bismark on Mox on a series of subset of the reads:
Methylation Analysis - O.lurida Bismark on Mox
This is a quick and dirty (i.e. no adaptor/quality trimming) assessment of all of our Ostrea lurida bisulfite sequencing efforts to date in order to get a rough idea of the methylation mapping, per this GitHub issue. Ran Bismark on Mox on a series of subset of the reads:
Methylation Analysis - C.virginica
This is a quick and dirty (i.e. no adaptor/quality trimming) assessment of all of our Crassostrea irginica bisulfite sequencing efforts to date in order to get a rough idea of the methylation mapping, per this GitHub issue. Ran Bismark on Mox on a series of subset of the reads:
Methylation Analysis - C.gigas Bismark on Mox
This is a quick and dirty (i.e. no adaptor/quality trimming) assessment of all of our Crassostrea gigas bisulfite sequencing efforts to date in order to get a rough idea of the methylation mapping, per this GitHub issue. Ran Bismark on Mox on a series of subset of the reads:
Data Management - Create C.virginica Bisulfite Genome wit Bismark on Mox
In preparation for some a quick and “dirty” bisulfite analysis, I needed to make bisulfite genomes with, and for, Bismark to use.
Data Management - Create C.gigas Bisulfite Genome with Bismark on Mox
In preparation for some a quick and “dirty” bisulfite analysis, I needed to make bisulfite genomes with, and for, Bismark to use.
Data Management - Create Geoduck Bisulfite Genomes with Bismark on Mox
In preparation for some a quick and “dirty” bisulfite analysis, I needed to make bisulfite genomes with, and for, Bismark to use.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Juvenile Day 5
Based on a discussion in this GitHub Issue, I’ve initiated some tissue-specific transcriptome assemblies with our current geoduck data.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Heart
Based on a discussion in this GitHub Issue, I’ve initiated some tissue-specific transcriptome assemblies with our current geoduck data.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Gonad
Based on a discussion in this GitHub Issue, I’ve initiated some tissue-specific transcriptome assemblies with our current geoduck data.
Transcriptome Assembly - Geoduck Tissue-specific Assembly Ctenidia
Based on a discussion in this GitHub Issue, I’ve initiated some tissue-specific transcriptome assemblies with our current geoduck data.
Genome Annotation - Pgenerosa_v71 with MAKER on Mox
With the P.generosa v070 annotation taking a very long time (due to the genome/transcriptome sizes, and, hitting some snags in the script I put together), Steven asked if I could subset the genome and annotate the v071 assembly (>10kbp subset) in order to have some annotations to use for some talks coming up ASLO (in this GitHub issue).
Sequence Subsetting - Pgenerosa_v70 Genome Assembly with faidx
Steven asked to subset geoduck assembly in to >10kbp and >30kbp groups. Here are the commands, using faidx:
DNA Isolation and Quantification - Ronit’s C.gigas Ploidy Ctenidia
Last week, I isolated DNA from all of Ronti’s ctenidia samples, however one sample (D18-C) didn’t isolate properly. So, I performed another isolation procedure with another section of frozen tissue. Tissue was excised from frozen tissue block via razor blade (weight not recorded) and pulverized under liquid nitrogen. Samples were incubated O/N @ 37oC (heating block) in 350uL of MB1 Buffer + 25uL Proteinase K, per the E.Z.N.A. Mollusc DNA Kit (Omega) instructions.
DNA Isolation - C.gigas Ploidy Experiment Ctenidia
Isolated DNA from the remaining ctenidia tissue samples from Ronit’s experiment (Google Sheet).
Gene Prediction - HiSeqX Metagenomics from Geoduck Water Using MetaGeneMark on Mox
After assembline the metagenomic data yesterday, I needed to predict some genes. I did this using MetaGeneMark (v.3.38) and ran it on Mox.
Metagenome Assembly - P.generosa Water Sample Trimmed HiSeqX Data Using Megahit on Mox
Attempting an assembly of our geoduck metagenomics HiSeqX data using Megahit (v.1.1.4) on a Mox node.
VCF Splitting - C.virginica VCF Using BCFtools
DNA Isolation - C.gigas Ploidy Experiment Ctenidia
Yesterday, Ronit initiated DNA isolation from ctenidia samples from his experiment (Google Sheet) from the following four samples:
qPCR - Relative mitochondrial abundance in C.gigas diploids and triploids subjected to acute heat stress via COX1
Using the C.gigas cytochrome c oxidase (COX1) primers (SR IDs: 1713, 1714)I designed the other day, I ran a qPCR on a subset of Ronit’s diploid/triploid control/heat shocked oyster DNA that Shelly had previously isolated and performed global DNA methylation assay. The goal is to get a rough assessment of whether or not there appear to be differences in relative mitochondrial abundances between these samples.
BLASTx - Clupea pallasii (Pacific herring) liver and testes transcriptomes on Mox
Apparently we’ve had some interest in the Pacific herring transcriptomes (liver and testes) we produced many years ago. As such, Steven asked that I do a quick BLASTx to help annotate these two transcriptomes.
FastQC and Trimming - Metagenomics (Geoduck) HiSeqX Reads from 20180809
Steven tasked me with assembling our geoduck metagenomics HiSeqX data. The first part of the process is examining the quality of the sequencing reads, performing quality trimming, and then checking the quality of the trimmed reads. It’s also possible (likely) that I’ll need to run another round of trimming. The process is documented in the Jupyter Notebook linked below. After these reads are cleaned up, I’ll transfer them over to our HPC nodes (Mox) and try assembling them.
Primer Design - Gigas COX1 using Primer3
We’re attempting a quick & dirty comparison of relative mitochondria counts between diploid and triploid C.gigas, so needed a single-copy mitochondrial gene target for qPCR. Selected cytochrome c oxidase subunit 1 (COX1), based on a quick glance at the NCBI mt genome browser for C.gigas NC_001276.
qPCR - Geoduck gonad cDNA with vitellogenin primers
Earlier today I made some cDNA from geoduck gonad RNA for use in this qPCR to test out the vitellogenin primers I designed on 20181129
Reverse Transcription - Geoduck gonad RNA pool
To be able to actually test the vitellegenin primers I made, I needed some geoduck cDNA.
Primer Design - Geoduck Vitellogenin using Primer3
In preparation for designing primers for developing a geoduck vitellogenin qPCR assay, I annotated a de novo geoduck transcriptome assembly last week. Next up, identify vitellogenin genes, design primers, confirm their specificity, and order them!
Annotation - Geoduck Transcritpome with TransDecoder
I was tasked with generating some qPCR primers to analyze vitellogenin expression in geoduck. In order to do so, I needed to annotate a geoduck transcriptome in order to identify potential vitellogenin genes. I had previously assembled a geoduck transcriptome. For annotation, I used TransDecoder (v5.5.0). The annotation was run on our Mox HPC node.
Reverse Transcription - Ronit’s C.gigas DNased RNA from 20181115
Quantified Ronit’s DNased RNA earlier today and proceeded with reverse transcription using 100ng of DNased RNA with oligo dT primers (Promega) and M-MLV reverse transcriptase (Promega) according to the manufacturer’s protocol.
RNA Quantification - Ronit’s C.gigas DNased RNA from 20181115
After confirming Ronit’s DNased RNA was free of gDNA, I quantified the DNased RNA from 20181115 using the Roberts Lab Qubit 3.0 and the Qubit hsRNA Assay.
qPCR - Ronit’s DNased C.gigas RNA with Elongation Factor Primers
After DNasing Ronit’s RNA earlier today, I needed to verify the RNA was free of any contaminating gDNA.
DNase - Ronit’s C.gigas Ctenidia RNA
DNA Isolation and Quantification - Lotterhos C.virginica Mantle DNA
Isolated DNA from the Lotterhos Crassostrea virginica mantle samples received on 20181017 using the E.Z.N.A. Mollusc Kit, with the following modifications/notes:
Computing - Install NCBI nr nt BLAST Database on Mox
Per this issue on GitHub, I installed the pre-formatted NCBI non-redudant (nr) nucleotide (nt) database on Mox.
qPCRs - Ronit’s C.gigas ploidy/dessication/heat stress cDNA (1:5 dilution)
IMPORTANT: The cDNA used for the qPCRs described below was a 1:5 dilution of Ronit’s cDNA made 20181017 with the following primers! Diluted cDNA was stored in his -20oC box with his original cDNA.
Reverse Transcription - Ronit’s C.gigas DNased ctenidia RNA
Proceeded with reverse transcription of [Ronit’s DNased ctenidia RNA (from 20181016)(2018-10-16-dnase-treatment-ronits-c-gigas-ploiyddessication-ctenidia-rna.html).
qPCR - Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with elongation factor primers
After I figured out the appropriate DNA and primers to use to detect gDNA in Crassostrea gigas samples, I checked Ronit’s [DNased ctenidia RNA (from 20181016)(2018-10-16-dnase-treatment-ronits-c-gigas-ploiyddessication-ctenidia-rna.html) for residual gDNA.
qPCR – C.gigas primer and gDNA tests with 18s and EF1 primers
The qPCR I ran earlier today to check for residual gDNA in Ronit’s DNased RNA turned out terribly, due to a combination of bad primers and, possibly, bad gDNA.
qPCR - Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with 18s primers
After DNasing Ronit’s RNA earlier today, I needed to check for any residual gDNA.
DNase Treatment - Ronit’s C.gigas Ploiyd/Dessication Ctenidia RNA
After quantifying Ronit’s RNA earlier today, I DNased them using the Turbo DNA-free Kit (Ambion), according to the manufacturer’s standard protocol.
RNA Quantification - Ronit’s C.gigas Ploidy/Dessication RNA
Last Friday, Ronit quantified 1:10 dilutions of the RNA I isolated on 20181003 and the RNA he finished isolating on 20181011, but two of the samples (D11-C, T10-C) were still too concentrated.
Data Received - Chionoecetes bairdi RNAseq & FastQC Analysis
We received Grace’s 100bp PE NovaSeq (Illumian) RNAseq data from the Northwest Genomics Center today.
VCF Splitting with bcftools
RNA Isolation - Tanner Crab Hemolymph Pellet in RNAlater using TriReagent
I previously isolated RNA from crab hemolymp from a lyophilized sample using TriReagent and Grace recently tried isolating RNA from crab hemolyph pellet (non-lyophilized) using TriReagent. The results for her extractions weren’t so great, so I’m giving it a shot with the following samples:
RNA Isolation - Ronit’s C.gigas diploid/triploid dessication/heat shock ctenidia tissues
Isolated RNA from a subset of Ronit’s Crassostrea gigas ctenidia samples (see Ronit’s notebook for experiment deets):
Installation - Microsoft Machine Learning Server (Microsoft R Open) on Emu/Roadrunner R Studio Server
Steven recently saw an announcement that Microsoft R Open now handles multi-threaded processing (default R does not), so we were interested in trying it out. I installed MLR/MRO on Emu/Roadrunner (Apple Xserve; Ubuntu 16.04). Followed the Microsoft installation directions for Ubuntu. In retrospect, I think I could’ve just installed MRO, but this gets the job done as well and won’t hurt anything.
Transcriptome Alignment & Bedgraph – Olympia oyster transcriptome with Olurida_v080 genome assembly
Bedgraph – Olympia oyster transcriptome with Olurida_v081 genome assembly
I took the sorted BAM file from yesterday’s corrected RNAseq genome alignment and converted it to a bedgraph using BEDTools genomeCoverageBed tool.
Transcriptome Alignment – Olympia oyster RNAseq reads aligned to genome with HISAT2
Yesterday’s attempt at producing a bedgraph was a failure and a prodcuct of a major brain fart. The worst part is that I was questioning what I was doing the entire time, but still went through with the process! Yeesh!
Bedgraph - Olympia oyster transcriptome (FAIL)
Progress on generating bedgraphs from our Olympia oyster transcriptome continues.
Transcriptome Alignment - Olympia oyster Trinity transcriptome aligned to genome with Bowtie2
Progress on generating bedgraphs from our Olympia oyster transcriptome continues.
Transcriptome Assembly - Olympia oyster RNAseq Data with Trinity
Used all of our current oly RNAseq data to assemble a transcriptome using Trinity.
DNA Methylation Analysis – Olympia Oyster Whole Genome BSseq Bismark Pipeline MethylKit Comparison
I previously ran two variations on the Bismark analysis for our Olympia oyster whole genome bisulfite sequencing data:
RNA Isolation - Lyophilized Tanner Crab Hemolymph in RNAlater
Due to difficulties getting RNA from hemolymph samples stored in RNAlater, Grace is testing out lyophilizing samples before extraction. Who knows what impact this will have on RNA, but it’s worth a shot!
DNA Methylation Analysis - Olympia Oyster Whole Genome BSseq Bismark Pipeline Comparison
Ran Bismark using our high performance computing (HPC) node, Mox, with two different bowtie2 settings:
DNA Quantification - Sea Lice DNA from 20180523
We previously received sea lice (Caligus tape) DNA from Cris Gallardo-Escarate at Universidad de Concepción.
DNA Methylation Analysis - Olympia oyster BSseq MethylKit Analysis
NOTE: IMPORTANT CAVEATS - READ POST BEFORE USING DATA.
Transcriptome Assembly - Geoduck RNAseq data
Used all of our current geoduck RNAseq data to assemble a transcriptome using Trinity.
FastQC/MultiQC/TrimGalore/MultiQC/FastQC/MultiQC - O.lurida WGBSseq for Methylation Analysis
I previously ran this data through the Bismark pipeline and followed up with MethylKit analysis. MethylKit analysis revealed an extremely low number of differentially methylated loci (DML), which seemed odd.
Transposable Element Mapping – Crassostrea virginica Genome, Cvirginica_v300, using RepeatMasker 4.07
Per this GitHub issue, I’m IDing transposable elements (TEs) in the Crassostrea virginica genome.
DNA Methylation Analysis - Bismark Pipeline on All Olympia oyster BSseq Datasets
Bismark analysis of all of our current Olympia oyster (Ostrea lurida) DNA methylation high-throughput sequencing data.
Data Received - Geoduck Metagenome HiSeqX Data
Received the data from the geoduck metagenome libraries that I prepared and were sequenced at the Northwest Genomics Center at UW on the HiSeqX (Illumina) - PE 151bp.
RNA Isolation & Quantificaiton - Tanner Crab Hemolymph
Isolated RNA from 40 Tanner crab hemolymph samples selected by Grace with the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s protocol, with the following modifications:
Bioanalyzer - Tanner Crab RNA Isolated with RNeasy Plus Mini Kit
Ran the four Tanner crab RNA samples that I isolated yesterday on the Seeb Lab Bioanalyzer 2100 (Agilent) using the RNA Pico 6000 Kit.
RNA Cleanup - Tanner Crab RNA
In a continued attempt to figure out what we can do about the tanner crab RNA, Steven tasked me with using an RNeasy Kit to cleanup some existing RNA.
RNA Isolation - Tanner Crab Hemolymph Using RNeasy Plus Mini Kit
Tanner crab RNA has proved a bit troublesome. As such, [Steven asked me to try isolating some RNA using the RNeasy Plus Mini Kit (Qiagen)(https://github.com/RobertsLab/resources/issues/327) to see how things would turn out.
Mox - Password-less SSH!
The high performance computing (HPC) cluster (called Mox) at Univ. of Washington (UW) frustratingly requires a password when SSH-ing, even when SSH keys are in use. I have a lengthy, unintelligible password that I use for my UW account, so having to type this in any time I want to initiate a new SSH session on Mox is a painful process.
Transposable Element Mapping – Olympia Oyster Genome Assembly, Olurida_v081, using RepeatMasker 4.07
I previously performed this analysis using a different version of our Ostrea lurida genome assembly. [Steven asked that I repeat the analysis with a modified version of the genome assembly (Olurida_v081)(https://github.com/RobertsLab/resources/issues/265#issuecomment-401055771) - only has contigs >1000bp in length.
Library Construction - Geoduck Water Filter Metagenome with Nextera DNA Flex Kit (Illumina)
Made Illumina libraries with goeduck metagenome water filter DNA I previously isolated on:
BS-seq Mapping – Olympia oyster bisulfite sequencing: Bismark Continued
Transposable Element Mapping – Crassostrea virginica NCBI Genome Assembly using RepeatMasker 4.07
Genome used: NCBI GCA_002022765.4_C_virginica-3.0
Read Mapping – Olympia oyster 2bRAD Data with Bowtie2 (on Mox)
Transposable Element Mapping - Olympia Oyster Genome Assembly using RepeatMasker 4.07
Software Installation – RepeatMasker v4.0.7 on Emu/Roadrunner Continued
After yesterday’s difficulties getting RMblast to compile, I deleted the folder and went through the build process again.
Software Installation - RepeatMasker v4.0.7 on Emu/Roadrunner
Steven asked that I re-run some Olympia oyster transposable elements analysis using RepeatMasker and a newer version of our Olympia oyster genome assembly.
TrimGalore/FastQC/MultiQC – TrimGalore! RRBS Geoduck BS-seq FASTQ data (directional)
Earlier this week, I ran TrimGalore!, but set the trimming, incorrectly - due to a copy/paste mistake, as --non-directional, so I re-ran with the correct settings.
FastQC - RRBS Geoduck BS-seq FASTQ data
Earlier today I finished trimming Hollie’s RRBS BS-seq FastQ data.
TrimGalore/FastQC/MultiQC – TrimGalore! RRBS Geoduck BS-seq FASTQ data
DNA Isolation & Quantification - Metagenomics Water Filters
After discussing the preliminary DNA isolation attemp with Steven & Emma, we decided to proceed with DNA isolations on the remaining 0.22μm filters.
Total Alkalinity Calculations - Yaamini’s Ocean Chemistry Samples
I ran a subset of Yaamini’s ocean chemistry samples on our T5 Excellence titrator (Mettler Toledo) at the beginning of April. The subset were samples taken from the beginning, middle, and end of the experiment. The rationale for this was to assess whether or not total alkalinity (TA) varied across the experiment. If there was little variation, then there’d likely be no need to run all of the samples. However, should there be temporal differences, then all samples should be processed.
TrimGalore/FastQC/MultiQC - Trim 10bp 5’/3’ ends C.virginica MBD BS-seq FASTQ data
Steven found out that the Bismarck documentation (Bismarck is the bisulfite aligner we use in our BS-seq pipeline) suggests trimming 10bp from both the 5’ and 3’ ends. Since this is the next step in our pipeline, we figured we should probably just follow their recommendations!
DNA Isolation & Quantification - Metagenomics Water Filters
Isolated DNA from the following two filters:
TrimGalore/FastQC/MultiQC - 2bp 3’ end Read 1s Trim C.virginica MBD BS-seq FASTQ data
Earlier today, I ran TrimGalore/FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch and hard trimmed the first 14bp from each read. Things looked better at the 5’ end, but the 3’ end of each of the READ1 seqs showed a wonky 2bp blip, so decided to trim that off.
TrimGalore/FastQC/MultiQC - 14bp Trim C.virginica MBD BS-seq FASTQ data
Yesterday, I ran TrimGalore/FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch with the default settings (i.e. “auto-trim”). There was still some variability in the first ~15bp of the reads and Steven wanted to see how a hard trim would change things.
TrimGalore/FastQC/MultiQC - Auto-trim C.virginica MBD BS-seq FASTQ data
Yesterday, I ran FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch. Steven wanted to trim it and see how things turned out.
FastQC/MultiQC - C. virginica MBD BS-seq Data
Per Steven’s GitHub Issues request, I ran FastQC on the Eastern oyster MBD bisulfite sequencing data we recently got back from ZymoResearch.
DNA Isolation & Quantification – Geoduck larvae metagenome filter rinses
This is another attempt to isolate DNA from two more of the geoduck hatchery metagenome samples Emma delivered on 20180313.
Titrations - Yaamini’s Seawater Samples
All data is deposited in the following GitHub repo:
Titrations - Yaamini’s Seawater Samples
All data is deposited in the following GitHub repo:
Data Received - Crassostrea virginica MBD BS-seq from ZymoResearch
Received the sequencing data from ZymoResearch for the Crassostrea virginica gonad MBD DNA that was sent to them on 20180207 for bisulfite conversion, library construction, and sequencing.
Titrations - Hollie’s Seawater Samples
All data is deposited in the following GitHub repo:
Titrations - Hollie’s Seawater Samples
All data is deposited in the following GitHub repo:
DNA Isolation & Quantification - Geoduck larvae metagenome filter rinses
Isolated DNA from two of the geoduck hatchery metagenome samples Emma delivered on 20180313 to get an idea of what type of yields we might get from these.
Titrations - Hollie’s Seawater Samples
Performed total alkalinity (TA) titrations on Hollie’s samples using our T5 Excellence titrator (Mettler Toledo) and Rondolino sample changer.
Titrations - Hollie’s Seawater Samples
Performed total alkalinity (TA) titrations on Hollie’s samples using our T5 Excellence titrator (Mettler Toledo) and Rondolino sample changer.
Progress Report - Titrator
I’ll begin this entry with a TL;DR (becuase it’s definitely a very long read):
Titrator Setup - Functional Methods & Data Exports
I’ve been working on getting our T5 Excellence titrator (Mettler Toledo) with Rondolino sample changer (Mettler Toledo) set up and operational.
Software Install - 10x Genomics Supernova on Mox (Hyak)
DNA Quantification - MspI-digested Crassostrea virginica gDNA
Quantified the two MspI-digested DNA samples for the Qiagen project from earlier today with the Qubit 3.0 (ThermoFisher).
Phenol:Chloroform Extractions and EtOH Precipitations - MspI Digestions of C.virginica DNA from Earlier Today
The two MspI restriction digestions from earlier today for our project with Qiagen were subjected to phenol:chloroform cleanup and subsequent ethanol precipitations.
Restriction Digestion - MspI on Crassotrea virginica gDNA
Digested two 1.5μg aliquots of Crassostrea virginica isolated 20171211, as part of the project we’re doing with Qiagen.
DNA Quantification - C.virginica MBD-enriched DNA
Quantified Crassostrea virginica MBD-enriched DNA from earlier today for Qiagen project.