Projects

qPCR - P.generosa APLP and TIF3s8-1 with cDNA

Shelly asked me to run some qPCRs (GitHub Issue), after some of the qPCR results I got from primer tests with normalzing genes and potential gene targets.

FastQ Read Alignment and Quantification - P.generosa Water Metagenomic Libraries to MetaGeneMark Assembly with Hisat2 on Mox

Continuing working on the manuscript for this data, Emma wanted the number of reads aligned to each gene. I previously created and assembly with genes/proteins using MetaGeneMark on 20190103, but the assemby process didn’t output any sort of stastics on read counts.

Primer Design and In-Silico Testing - Geoduck Reproduction Primers

Shelly asked that I re-run the primer design pipeline that Kaitlyn had previously run to design a set of reproduction-related qPCR primers. Unfortunately, Kaitlyn’s Jupyter Notebook wasn’t backed up and she accidentally deleted it, I believe, so there’s no real record of how she designed the primers. However, I do know that she was unable to run the EMBOSS primersearch tool, which will check your primers against a set of sequences for any other matches. This is useful for confirming specificity.

qPCR - Testing P.generosa Reproduction-related Primers

Ran some qPCRs on some other primers on 20200723 and then Shelly has asked me to test some additional qPCR primers that might have acceptable melt curves and be usable as normalizing genes.

SRA Submission - P.generosa Metagenomics Data

Added our P.generosa metagenomics sequencing data to NCBI sequencing read archive (SRA).

qPCR - Testing P.generosa Reproduction-related Primers

Shelly has asked me to test some qPCR primers related to geoduck reproduction.

DNA Isolation and Quantification - C.gigas Diploid (Ronit) and Triploid (Nisbet)

Isolated some gDNA from the triploid Nisbet oysters we received on 20200218 and one of Ronit’s diploid ctenidia samples (Google Sheet) using the E.Z.N.A. Mollusc DNA Kit (Omega). See the “Results” section for sample info.

Metagenomics - Data Extractions Using MEGAN6

Decided to finally take the time to methodically extract data from our metagenomics project so that I have the tables handy when I need them and I can easily share them with other people. Previously, I hadn’t done this due to limitations on looking at the data remotely. I finally downloaded all of the RMA6 files from 20191014 after being fed up with the remote desktop connection and upgrading the size of my hard drive (5 of the six RMA6 files are >40GB in size).

Transcriptome Annotation - C.bairdi Transcriptomes v2.1 and v3.1 Using DIAMOND BLASTx on Mox

Decided to annotate the two C.bairdi transcriptomes , cbai_transcriptome_v2.1 and cbai_transcriptome_v3.1, generated on 20200605 using DIAMOND BLASTx on Mox.

Transcriptome Assessment - BUSCO Metazoa on C.bairdi Transcriptome v3.1

Continuing to try to identify the best C.bairdi transcriptome, we decided to extract all non-dinoflagellate sequences from cbai_transcriptome_v2.0 (RNAseq shorthand: 2018, 2019, 2020-GW, 2020-UW) and cbai_transcriptome_v3.0 (RNAseq shorthand: 2018, 2019, 2020-UW).

Transcriptome Assessment - BUSCO Metazoa on C.bairdi Transcriptome v2.1

Continuing to try to identify the best C.bairdi transcriptome, we decided to extract all non-dinoflagellate sequences from cbai_transcriptome_v2.0 (RNAseq shorthand: 2018, 2019, 2020-GW, 2020-UW) and cbai_transcriptome_v3.0 (RNAseq shorthand: 2018, 2019, 2020-UW).

Sequence Extractions - C.bairdi Transcriptomes v2.0 and v3.0 Excluding Alveolata with MEGAN6 on Swoose

Continuing to try to identify the best C.bairdi transcriptome, we decided to extract all non-dinoflagellate sequences from cbai_transcriptome_v2.0 (RNAseq shorthand: 2018, 2019, 2020-GW, 2020-UW) and cbai_transcriptome_v3.0 (RNAseq shorthand: 2018, 2019, 2020-UW). Both of these transcriptomes were assembled without any taxonomic filter applied. DIAMOND BLASTx and conversion to MEGAN6 RMA6 files was performed yesterday (20200604).

Transcriptome Annotation - C.bairdi Transcriptomes v2.0 and v3.0 with DIAMOND BLASTx on Mox

Continuing to try to identify the best C.bairdi transcriptome, we decided to extract all non-dinoflagellate sequences from cbai_transcriptome_v2.0 (RNAseq shorthand: 2018, 2019, 2020-GW, 2020-UW) and cbai_transcriptome_v3.0 (RNAseq shorthand: 2018, 2019, 2020-UW). Both of these transcriptomes were assembled without any taxonomic filter applied.

Transcriptome Annotation - Trinotate C.bairdi Transcriptome-v1.7 on Mox

After creating a de novo assembly of C.bairdi transcriptome v1.7 on 20200527, performing BLASTx annotation on 202000527, and TransDecoder for ORF identification on 20200527, I continued the annotation process by running Trinotate.

Transcriptome Comparisons - C.bairdi BUSCO Scores

Since we’ve generated a number of versions of the C.bairdi transcriptome, we’ve decided to compare them using various metrics. Here, I’ve compared the BUSCO scores generated for each transcriptome using BUSCO’s built-in plotting script. The script generates a stacked bar plot of all BUSCO short summary files that it is provided with, as well as the R code used to generate the plot.

TransDecoder - C.bairdi Transcriptome v1.7 on Mox

Need to run TransDecoder on Mox on the C.bairdi transcriptome v1.7 from 20200527.

Transcriptome Annotation - C.bairdi Transcriptome v1.7 Using DIAMOND BLASTx on Mox

As part of annotating cbai_transcriptome_v1.7.fasta from 20200527, I need to run DIAMOND BLASTx to use with Trinotate.

Transcriptome Assessment - BUSCO Metazoa on C.bairdi Transcriptome v1.7

I previously created a C.bairdi de novo transcriptome assembly v1.7 with Trinity from all our C.bairdi taxonomically filtered pooled RNAseq samples on 20200527 and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.

Transcriptome Assembly - C.bairdi All Pooled Arthropoda-only RNAseq Data with Trinity on Mox

For completeness sake, I wanted to create an additional C.bairdi transcriptome assembly that consisted of Arthropoda only sequences from just pooled RNAseq data (since I recently generated a similar assembly without taxonomically filtered reads on 20200518). This constitutes samples we have designated: 2018, 2019, 2020-UW. A de novo assembly was run using Trinity on Mox. Since all pooled RNAseq libraries were stranded, I added this option to Trinity command.

Transcriptome Annotation - Trinotate C.bairdi Transcriptome-v3.0 on Mox

After performing de novo assembly on all of our Tanner crab RNAseq data (no taxonomic filter applied, either) on 20200518, I continued the annotation process by running Trinotate.

Transcriptome Assembly - P.trituberculatus (Japanese blue crab) NCBI SRA BioProject PRJNA597187 Data with Trinity on Mox

After generating a number of C.bairdi (Tanner crab) transcriptomes, we decided we should compare them to evaluate which to help decide which one should become our “canonical” version. As part of that, the Trinity wiki offers a list of tools that one can use to check the quality of transcriptome assemblies. Some of those require a transcriptome of a related species.

SRA Library Assessment - Determine RNAseq Library Strandedness from P.trituberculatus SRA BioProject PRJNA597187

We’ve produced a number of C.bairid transcriptomes utilizing different assembly approaches (e.g. Arthropoda reads only, stranded libraries only, mixed strandedness libraries, etc) and we want to determine which of them is “best”. Trinity has a nice list of tools to assess the quality of transcriptome assemblies, but most of the tools rely on comparison to a transcriptome of a related species.

Transcriptome Annotation - C.bairdi Transcriptome v1.6 Using DIAMOND BLASTx on Mox

As part of annotating cbai_transcriptome_v1.6.fasta from 20200518, I need to run DIAMOND BLASTx to use with Trinotate.

Transcriptome Assessment - BUSCO Metazoa on C.bairdi Transcriptome v1.6

I previously created a C.bairdi de novo transcriptome assembly v1.6 with Trinity from all our C.bairdi taxonomically filtered RNAseq on 20200518 and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.

Transcriptome Annotation - C.bairdi Transcriptome v3.0 Using DIAMOND BLASTx on Mox

As part of annotating cbai_transcriptome_v3.0.fasta from 20200518, I need to run DIAMOND BLASTx to use with Trinotate.

Transcriptome Assessment - BUSCO Metazoa on C.bairdi Transcriptome v3.0

I previously created a C.bairdi de novo transcriptome assembly with Trinity from all our C.bairdi pooled RNAseq (not taxonomically filtered) on 20200518 and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.

Transcriptome Assembly - C.bairdi All Arthropoda-specific RNAseq Data with Trinity on Mox

I realized I hadn’t performed taxonomic read separation from one set of RNAseq data we had. And, since I was on a transcriptome assembly kick, I figured I’d generate another C.bairdi transcriptome that included only Arthropoda-specific sequence data from all of our RNAseq.

Data Wrangling - Arthropoda and Alveolata D26 Pool 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.

Transcriptome Assembly - C.bairdi All Pooled RNAseq Data Without Taxonomic Filters with Trinity on Mox

Steven asked that I assemble a transcriptome with just our pooled C.bairdi RNAseq data (not taxonomically filtered; see the FastQ list file linked in the Results section below). This constitutes samples we have designated: 2018, 2019, 2020-UW. A de novo assembly was run using Trinity on Mox. Since all pooled RNAseq libraries were stranded, I added this option to Trinity command.

Transcriptome Annotation - Trinotate C.bairdi Transcriptome v2.0 from 20200502 on Mox

After performing de novo assembly on all of our Tanner crab RNAseq data (no taxonomic filter applied, either) on 20200502 and performing BLASTx annotation on 20200508, I continued the annotation process by running Trinotate.

TransDecoder - C.bairdi Transcriptome v2.0 from 20200502 on Mox

Need to run TransDecoder on Mox on the C.bairdi transcriptome v2.0 from 20200502.

Transcriptome Annotation - C.bairdi Transcriptome v2.0 Using DIAMOND BLASTx on Mox

As part of annotating the C.bairdi v2.0 transcriptome assembly from 20200502, I need to run DIAMOND BLASTx to use with Trinotate.

Transcriptome Assessment - BUSCO Metazoa on C.bairdi v2.0 Transcriptome

I previously created a C.bairdi de novo transcriptome assembly with Trinity using all existing, unfiltered (i.e. no taxonomic selection) RNAseq data on 20200502 and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.

Transcriptome Assembly - C.bairdi All RNAseq Data Without Taxonomic Filters with Trinity on Mox

Steven asked that I assemble an unfiltered (i.e. no taxonomic selection) transcriptome with all of our C.bairdi RNAseq data (see the FastQ list file linked in the Results section below). A de novo assembly was run using Trinity on Mox. It should be noted that this assembly is a mixture of stranded/non-stranded library preps.

GO to GOslim - C.bairdi Enriched GO Terms from 20200422 DEGs

After running pairwise comparisons and identify differentially expressed genes (DEGs) on 20200422 and finding enriched gene ontology terms, I decided to map the GO terms to Biological Process GOslims. Additionally, I decided to try another level of comparison (I’m not sure how valid it is), whereby I will count the number of GO terms assigned to each GOslim and then calculate the percentage of GOterms that get assigned to each of the GOslim categories. The idea being that it might help identify Biological Processes that are “favored” in a given set of DEGs. I decided to set up “fancy” pyramid plots to view a given set of GO-GOslims for each DEG comparison.

FastQC-MultiQC - Laura Spencer’s QuantSeq Data

Laura Spencer received her O.lurida QuantSeq data, so I put it through FastQC/MultiQC and put the pertinent info in the nightingales Google Sheet. I also moved the data to /owl/nightingales/O_lurida, updated the readme file and checksums file. There were 148 individual samples, so I won’t list them all here.

Gene Expression - C.bairdi Pairwise DEG Comparisons with 2019 RNAseq using Trinity-Salmon-EdgeR on Mox

Per a Slack request, Steven asked me to take the Genewize RNAseq data (received 2020318) through edgeR. Ran the analysis using the Trinity differential expression pipeline:

RNAseq Reads Extractions - C.bairdi Taxonomic Reads Extractions with MEGAN6 on swoose

I previously annotated reads and converted them to the MEGAN6 format RMA6 on 20200414.

Transcript Abundance - C.bairdi Alignment-free with Salmon on Mox for Grace

Per this GitHub Issue, Grace and Steven asked if I could help by generating a transcript abundance file for Grace to use with EdgeR. To do so, I used Salmon for alignment-free transcript abundance estimates due to its speed and its incorporation into Trinity with the following files:

SRA Submission - C.bairdi RNAseq Data

Since we received the last of our RNAseq data for this project on 20200413, I submitted all of it to the NCBI Sequencing Read Archive (SRA). Data was released today and all accession numbers can be found in the table below:

TrimmingFastQCMultiQC—C.bairdi-RNAseq-FastQ-with-fastp-on-Mox

After receiving our RNAseq data from Genewiz earlier today, needed to trim and check trimmed reads with FastQC.

Taxonomic Assignments - C.bairdi RNAseq Using DIAMOND BLASTx on Mox and MEGAN6 Meganizer on swoose

After receiving/trimming the latest round of C.bairdi RNAseq data on 20200413, 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.

FastQC-MultiQC - C.bairdi Raw RNAseq from NWGSC

Yesterday, we received the last of the RNAseq data for the C.bairdi crab project from NWGSC. FastQC, followed by MultiQC was run on the raw FastQ reads on my computer (swoose).

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 20200330, 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.

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 20200330 and performing BLASTx annotation on 20200408, I continued the annotation process by running Trinotate.

Transcriptome Annotation - C.bairdi MEGAN Trinity Assembly Using DIAMOND BLASTx on Mox

As part of annotating the most recent transcriptome assembly from the MEGAN6 Arthropoda taxonomic-specific reads, I need to run DIAMOND BLASTx to use with Trinotate.

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 20200330 and performing BLASTx annotation on 20200331, I continued the annotation process by running Trinotate.

Transdecoder - Hematodinium MEGAN6 Taxonomic-Specific Reads Assembly from 20200330

Ran Trinity to de novo assembly on the the Alveolata MEGAN6 taxonomic-specific RNAseq data on 20120330 and now will begin annotating the transcriptome using TransDecoder on Mox.

Transdecoder - C.bairdi MEGAN6 Taxonomic-Specific Reads Assembly from 20200330

Ran Trinity to de novo assembly on the the Arthropoda MEGAN6 taxonomic-specific RNAseq data on 20120330 and now will begin annotating the transcriptome using TransDecoder on Mox.

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

I previously annotated reads and converted them to the MEGAN6 format RMA6 on 20200318.

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 (for reference, these include RNAseq data using a newly established “shorthand”: 2018, 2019), 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 (for reference, these include RNAseq data using a newly established “shorthand”: 2018, 2019). 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 (for reference, these include RNAseq data using a newly established “shorthand”: 2018, 2019) 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

We recently had a meeting with Emma and Brook about the progress of this metagenomics project and Brook suggested trying out MEGAN6 as user-friendly way to get these samples annotated.

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”.