Posts by Year

2021

Genome Assembly - Olurida_v090 with BGI Illumina and PacBio Hybrid Using Wengan on Mox

  • 3 min read

I was recently tasked with adding annotations for our Ostrea lurida genome assembly to NCBI. As it turns out, adding just annotation files can’t be done since the genome was initially submitted to ENA. Additionally, updating the existing ENA submission with annotations is not possible, as it requires a revocation of the existing genome assembly; requiring a brand new submission. With that being the case, I figured I’d just make a new genome submission with the annotations to NCBI. Unfortunately, there were a number of issues with our genome assembly that were going to require a fair amount of work to resolve. The primary concern was that most of the sequences are considered “low quality” by NCBI (too many and too long stretches of Ns in the sequences). Revising the assembly to make it compatible with the NCBI requirements was going to be too much, so that was abandoned.

Read More

Trimming - O.lurida BGI FastQs with FastP on Mox

  • 3 min read

After attempting to submit our Ostrea lurida (Olympia oyster) genome assembly annotations (via GFF) to NCBI, the submission process also highlighted some short comings of the Olurida_v081 assembly. When getting ready to submit the genome annotations to NCBI, I was required to calculate the genome coverage we had. NCBI suggested to calculate this simply by counting the number of bases sequenced and divide it by the genome size. Doing this resulted in an estimated coverage of ~55X coverage, yet we have significant stretches of Ns throughout the assembly. I understand why this is still technically possible, but it’s just sticking in my craw. So, I’ve decided to set up a quick assembly to see what I can come up with. Of note, the canonical assembly we’ve been using relied on the scaffolded assembly provided by BGI; we never attempted our own assembly from the raw data.

Read More

Genome Submission - Validation of Olurida_v081.fa and Annotated GFFs Prior to Submission to NCBI

  • 3 min read

Per this GitHub Issue, Steven has asked to get our Ostrea lurida (Olympia oyster) genome assembly (Olurida_v081.fa) submitted to NCBI with annotations. The first step in the submission process is to use the NCBI table2asn_GFF software to validate the FastA assembly, as well as the GFF annotations file. Once the software has been run, it will point out any errors which need to be corrected prior to submission.

Read More

Read Mapping - 10x-Genomics Trimmed FastQ Mapped to P.generosa v1.0 Assembly Using Minimap2 for BlobToolKit on Mox

  • 2 min read

To continue towards getting our Panopea generosa (Pacific geoduck) genome assembly (v1.0) analyzed with BlobToolKit, per this GitHub Issue, I’ve decided to run each aspect of the pipeline manually, as I continue to have issues utilizing the automatic pipeline. As such, I’ve run minimap2 according to the BlobToolKit “Getting Started” guide on Mox. This will map the trimmed 10x-Genomics reads from 20210401 to the Panopea-generosa-v1.0.fa assembly (FastA; 914MB).

Read More

TransDecoder - C.bairdi Transcriptome v4.0 on Mox

  • 1 min read

Began annotation of cbai_transcriptome_v4.0.fasta [Trinity de novo assembly from 20210317(https://robertslab.github.io/sams-notebook/2021/03/17/Transcriptome-Assembly-C.bairdi-Transcriptome-v4.0-Using-Trinity-on-Mox.html)] using TransDecoder on Mox. This will be used as a component of Trinotate annotation downstream.

Read More

Back to Top ↑

2020

Transcriptome Comparisons - C.bairdi Transcriptomes Evaluations with DETONATE rsem-eval on Mox

  • 5 min read

UPDATE: I’ll lead in with the fact that this failed with an error message that I can’t figure out. This will save the reader some time. I’ve posted the problem as an Issue on the DETONATE GitHub repo, however it’s clear that this software is no longer maintained, as the repo hasn’t been updated in >3yrs; even lacking responses to Issues that are that old.

Read More

Alignments - C.bairdi RNAseq Transcriptome Alignments Using Bowtie2 on Mox

  • 5 min read

I had previously attempted to compare all of our C.bairdi transcriptome assemblies using DETONATE on 20200601, but, due to hitting time limits on Mox, failed to successfully get the analysis to complete. I realized that the limiting factor was performing FastQ alignments, so I decided to run this step independently to see if I could at least get that step resolved. DETONATE (rsem-eval) will accept BAM files as input, so I’m hoping I can power through this alignment step and then provided DETONATE (rsem-eval) with the BAM files.

Read More

FastQC-MultiQc - C.gigas Ploidy pH WGBS Raw Sequence Data from Haws Lab on Mox

  • 2 min read

Yesterday (20201205), we received the whole genome bisulfite sequencing (WGBS) data back from ZymoResearch from the 24 C.gigas diploid/triploid subjected to two different pH treatments (received from the Haws’ Lab on 20200820 that we submitted to ZymoResearch on 20200824. As part of our standard sequencing data receipt pipeline, I needed to generate FastQC files for each sample.

Read More

Sample Submission - M.magister MBD BSseq Libraries for MiSeq at NOAA

  • 1 min read

Earlier today I quantified the libraries with the Qubit in preparation for sample pooling and sequencing. Before performing a full sequencing run, Mac wanted to select a subset of the libraries based on the experimental treatments to have an equal representation of samples. She also wanted to do a quick run on the MiSeq at NOAA to evaluate how well libraries map and to make sure libraries appear to be sequencing at relatively equal levels.

Read More

Transcriptome Assessment - Crustacean Transcripome Completeness Evaluation Using BUSCO on Mox

  • 4 min read

Grace was recently working on writing up a manuscript which did a basic comparison of our C.bairdi transcriptome (cbai_transcriptome_v3.1) (see the Genomic Resources wiki for more deets) to two other species’ transcriptome assemblies. We wanted BUSCO evaluations as part of this comparison, but the two other species did not have BUSCO scores in their respective publications. As such, I decided to generate them myself, as BUSCO runs very quickly. The job was run on Mox.

Read More

Hard Drive Upgrade - Gannet Synology Server

  • ~1 min read

Completed upgrading the 12 x 8TB HDDs in our server, Gannet (Synology RS3618XS), to 12 x 16TB HDDs. The process was simple, but the repair process took ~20hrs for each new drive. So, the entire process required 12 separate days of pulling out one old HDD, replacing with a new HDD, and initiating the repair process in the Synology web interface.

Read More

MBD Selection - M.magister Sheared Gill gDNA 16 of 24 Samples Set 3 of 3

  • 1 min read

Click here for notebook on the first eight samples processed. Click here for the second set of eight samples processed. M.magister (Dungeness crab) gill gDNA provided by Mackenzie Gavery was previously sheared on 20201026 and three samples were subjected to additional rounds of shearing on 20201027, in preparation for methyl bidning domain (MBD) selection using the MethylMiner Kit (Invitrogen).

Read More

Trimming - Shelly S.salar RNAseq Using fastp and MultiQC on Mox

  • 3 min read

Shelly asked that I trim, align to a genome, and perform transcriptome alignment counts in this GitHub issue with some Salmo salar RNAseq data she had and, using a subset of the NCBI Salmo salar RefSeq genome, GCF_000233375.1. She created a subset of this genome using only sequences designated as “chromosomes.” A link to the FastA (and a link to her notebook on creating this file) are in that GitHub issue link above. The transcriptome she has provided has not been subsetted in a similar fashion; maybe I’ll do that prior to alignment.

Read More

DNA Shearing - M.magister CH05-21 gDNA Full Shearing Test and Bioanalyzer

  • 2 min read

Yesterday, I did some shearing of Metacarcinus magister gill gDNA on a test sample (CH05-21) to determine how many cycles to run on the sonicator (Bioruptor 300; Diagenode) to achieve an average fragment length of ~350 - 500bp in preparation for MBD-BSseq. The determination from yesterday was 70 cycles (30s ON, 30s OFF; low intensity). That determination was made by first sonicating for 35 cycles, followed by successive rounds of 5 cycles each. I decided to repeat this, except by doing it in a single round of sonication.

Read More

DNA Shearing - M.magister gDNA Shear Testing and Bioanalyzer

  • 1 min read

Steven assigned me to do some MBD-BSseq library prep (GitHub Issue) for some Dungeness crab (Metacarcinus magister) DNA samples provided by Mackenzie Gavery. The DNA was isolated from juvenile (J6/J7 developmental stages) gill tissue. One of the first steps in MBD-BSseq is to fragment DNA to a desired size (~350 - 500bp in our case). However, we haven’t worked with Metacarcinus magister DNA previously, so I need to empirically determine sonicator (Bioruptor 300; Diagenode) settings for these samples.

Read More

Read Mapping - C.bairdi 201002558-2729-Q7 and 6129-403-26-Q7 Taxa-Specific NanoPore Reads to cbai_genome_v1.01.fasta Using Minimap2 on Mox

  • 2 min read

After extracting FastQ reads using seqtk on 20201013 from the various taxa I had been interested in, the next thing needed doing was mapping reads to the cbai_genome_v1.01 “genome” assembly from 20200917. I found that Minimap2 will map long reads (e.g. NanoPore), in addition to short reads, so I decided to give that a rip.

Read More

Data Wrangling - C.bairdi NanoPore Reads Extractions With Seqtk on Mephisto

  • 1 min read

In my pursuit to identify which contigs/scaffolds of our C.bairdi” genome assembly from 20200917 correspond to interesting taxa, based on taxonomic assignments produced by MEGAN6 on 20200928, I used MEGAN6 to extract taxa-specific reads from cbai_genome_v1.01 on 20201007 - the output is only available in FastA format. Since I want the original reads in FastQ format, I will use the FastA sequence IDs (from the FastA index file) and provide that to seqtk to extract the FastQ reads for each sample and corresponding taxa.

Read More

Taxonomic Assignments - C.bairdi 6129-403-26-Q7 NanoPore Reads Using DIAMOND BLASTx on Mox and MEGAN6 daa2rma on emu

  • 3 min read

After noticing that the initial MEGAN6 taxonomic assignments for our combined C.bairdi NanoPore data from 20200917 revealed a high number of bases assigned to E.canceri and Aquifex sp., I decided to explore the taxonomic breakdown of just the individual samples to see which of the samples was contributing to these taxonomic assignments most.

Read More

Taxonomic Assignments - C.bairdi 20102558-2729-Q7 NanoPore Reads Using DIAMOND BLASTx on Mox and MEGAN6 daa2rma on emu

  • 3 min read

After noticing that the initial MEGAN6 taxonomic assignments for our combined C.bairdi NanoPore data from 20200917 revealed a high number of bases assigned to E.canceri and Aquifex sp., I decided to explore the taxonomic breakdown of just the individual samples to see which of the samples was contributing to these taxonomic assignments most.

Read More

Data Wrangling - C.bairdi NanoPore 6129-403-26 Quality Filtering Using NanoFilt on Mox

  • 2 min read

Last week, I ran all of our Q7-filtered C.baird NanoPore reads through MEGAN6 to evaluate the taxonomic breakdown (on 20200917) and noticed that there were a large quantity of bases assigned to E.canceri (a known microsporidian agent of infection in crabs) and Aquifex sp. (a genus of thermophylic bacteria), in addition to the expected Arthropoda assignments. Notably, Alveolata assignments were remarkably low.

Read More

Data Wrangling - C.bairdi NanoPore 20102558-2729 Quality Filtering Using NanoFilt on Mox

  • 2 min read

Last week, I ran all of our Q7-filtered C.baird NanoPore reads through MEGAN6 to evaluate the taxonomic breakdown (on 20200917) and noticed that there were a large quantity of bases assigned to E.canceri (a known microsporidian agent of infection in crabs) and Aquifex sp. (a genus of thermophylic bacteria), in addition to the expected Arthropoda assignments. Notably, Alveolata assignments were remarkably low.

Read More

Data Wrangling - Subsetting cbai_genome_v1.0 Assembly with faidx

  • 1 min read

Previously assembled cbai_genome_v1.0.fasta with our NanoPore Q7 reads on 20200917 and noticed that there were numerous sequences that were well shorter than the expected 500bp threshold that the assembler (Flye) was supposed to spit out. I created an Issue on the Flye GitHub page to find out why. The developer responded and determined it was an issue with the assembly polisher and that sequences <500bp could be safely ignored.

Read More

DNA Quantification - Re-quant Ronits C.gigas Diploid-Triploid Ctenidia gDNA Submitted to ZymoResearch

  • 1 min read

I received notice from ZymoResearch yesterday afternoon that the DNA we sent on 20200820 for this project (Quote 3534) had insufficient DNA for sequencing for most of the samples. This was, honestly, shocking. I had even submitted well over the minimum amount of DNA required (submitted 1.75ug - only needed 1ug). So, I’m not entirely sure what happened here.

Read More

Primer Design and In-Silico Testing - Geoduck Reproduction Primers

  • 1 min read

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.

Read More

Metagenomics - Data Extractions Using MEGAN6

  • 1 min read

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).

Read More

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

  • ~1 min read

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).

Read More

Transcriptome Comparison - C.bairdi Transcriptomes Compared with DETONATE on Mox

  • 4 min read

We’ve produced a number of C.bairdi transcriptomes and we’re interested in doing some comparisons to try to determine which one might be “best”. I previously compared the BUSCO scores of each of these transcriptomes and now will be using the DETONATE software package to perform two different types of comparisons: compared to a reference (REF-EVAL) and determine an overall quality “score” (RSEM-EVAL). I’ll be running REF-EVAL in this notebook.

Read More

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

  • 2 min read

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.

Read More

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

  • 3 min read

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.

Read More

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

  • 3 min read

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.

Read More

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

  • 2 min read

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.

Read More

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

  • 6 min read

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.

Read More

NanoPore Sequencing - C.bairdi gDNA 6129_403_26

  • 1 min read

After getting high quality gDNA from Hematodinium-infected C.bairdi hemolymph on 2020210 we decided to run some of the sample on the NanoPore MinION, since the flowcells have a very short shelf life. Additionally, the results from this will also help inform us on whether this sample might worth submitting for PacBio sequencing. And, of course, this provides us with additional sequencing data to complement our previous NanoPore runs from 20200109.

Read More

qPCR - C.bairdi RNA Check for Residual gDNA

  • 1 min read

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.

Read More

Trimming/MultiQC - Methcompare Bisulfite FastQs with fastp on Mox

  • 3 min read

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:

Read More

RNA Isolation and Quantification - C.bairdi RNA from Hemolymph Pellets in RNAlater

  • ~1 min read

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.

Read More

DNA Isolation, Quantification, and Gel - C.bairdi gDNA Sample 6129_403_26

  • 1 min read

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.

Read More

Gene Expression - Hematodinium MEGAN6 with Trinity and EdgeR

  • 2 min read

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.

Read More

Gene Expression - C.bairdi MEGAN6 with Trinity and EdgeR

  • 2 min read

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.

Read More

RNA Isolation and Quantification - C.bairdi Hemocyte Pellets in RNAlater Troubleshooting

  • 1 min read

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…

Read More

NanoPore Sequencing - Initial NanoPore MinION Lambda Sequencing Test

  • 1 min read

We recently acquired a NanoPore MinION sequencer, FLO-MIN106 flow cell and the Rapid Sequencing Kit (SQK-RAD004). The NanoPore website provides a pretty thorough an user-friendly walk-through of how to begin using the system for the first time. With that said, I believe the user needs to have a registered account with NanoPore and needs to have purchased some products to have full access to the protocols they provide.

Read More

Back to Top ↑

2019

Trimming/FastQC/MultiQC - C.bairdi RNAseq FastQ with fastp on Mox

  • 2 min read

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.

Read More

Data Wrangling - Rename Pgenerosa_v074 Files and Scaffolds

  • ~1 min read

Continuing to organizing files for a manuscript dealing with the geoduck genome assembly/annotation we’ve done, we decided to rename the files as well as rename the scaffolds, to make the naming consistent and a bit easier to read (both for humans and computers).

Read More

Data Wrangling - Splitting BAM by Size for Upload to OSF

  • ~1 min read

We’re in the process of organizing files for a manuscript dealing with the geoduck genome assembly/annotation we’ve done. As part of that, we need the Stringtie BAM file that was used with GenSAS for Pgenerosa_v074 annotation to upload to the Open Science Foundation repository for this project. Unfortunately, at 73GB, the file far exceeds the individual file size limit for OSF (5GB). So, I split it into 5GB chunks. See the following notebook for deets:

Read More

Data Wrangling - Panopea generosa Genome Feature Sequence Lengths

  • 1 min read

In preparation for a paper we’re writing, we needed some summary stats on the various genome assembly feature sequences. I determined the max/min, mean, and median sequence lengths for all the GFF feature files we currently have for Pgenerosa_v070, Pgenerosa_v070_top18_scaffolds, and Pgenerosa_v074. This info will be compiled in to a table for the manuscript. See our Genomic Resources wiki for more info on GFFs:

Read More

Genome Comparison - Pgenerosa_v074 vs Pgenerosa_v070 with MUMmer Promer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs S.glomerata NCBI with MUMmer Promer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs M.yessoensis NCBI with MUMmer Promer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs H.sapiens NCBI with MUMmer Promer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs C.virginica NCBI with MUMmer Promer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs C.gigas NCBI with MUMmer Promer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs Pgenerosa_v070 with MUMmer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs Pgenerosa_v074 with MUMmer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs S.glomerata NCBI with MUMmer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs M.yessoensis NCBI with MUMmer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs H.sapiens NCBI with MUMmer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs C.gigas NCBI with MUMmer on Mox

  • 3 min read

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.

Read More

Genome Comparison - Pgenerosa_v074 vs C.virginica NCBI with MUMmer on Mox

  • 3 min read

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.

Read More

Data Summary - P.generosa Transcriptome Assemblies Stats

  • 1 min read

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:

Read More

Genome Annotation - Pgenerosa_v074 Transcript Isoform ID with Stringtie on Mox

  • 4 min read

After annotating Pgenerosa_v070 and comparing feature counts, there was a drastic difference between the two genome versions. Additionally, both of those genomes ended up with no CDS/exon/gene/mRNA features identified in the largest scaffold. So, to explore this further by seeing where (if??) sequencing reads map to the scaffold, and to obtain transcript isoforms for the genome, I ran Stringtie. A Hisat2 index was prepared earlier.

Read More

Genome Annotation - Pgenerosa_v070 Transcript Isoform ID with Stringtie on Mox

  • 3 min read

After annotating Pgenerosa_v074 and comparing feature counts, there was a drastic difference between the two genome versions. Additionally, both of those genomes ended up with no CDS/exon/gene/mRNA features identified in the largest scaffold. So, to explore this further by seeing where (if??) sequencing reads map to the scaffold, and to obtain transcript isoforms for the genome, I ran Stringtie. A Hisat2 index was prepared earlier.

Read More

Genome Annotation - Pgenerosa_v070 and v074 Top 18 Scaffolds Feature Count Comparisons

  • 1 min read

After annotating Pgenerosa_v074 on 20190701, we noticed a large discrepancy in the number of transcripts that MAKER identified, compared to Pgenerosa_v070. As a reminder, the Pgenerosa_v074 is a subset of Pgenerosa_v070 containing only the top 18 longest scaffolds. So, we decided to do a quick comparison of the annotations present in these 18 scaffolds Pgenerosa_v070 and Pgenerosa_v074.

Read More

Genome Annotation - Pgenerosa_v071 Using GenSAS

  • 3 min read

In our various attempts to get the Panopea generosa genome annotated in such a manner that we’re comfortable with (the previous annotation attempts we’re lacking any annotations in almost all of the largest scaffolds, which didn’t seem right), Steven stumbled across GenSAS, a web/GUI-based genome annotation program, so we gave it a shot.

Read More

Genome Annotation - Pgenerosa_v074 Using GenSAS

  • 4 min read

In our various attempts to get the Panopea generosa genome annotated in such a manner that we’re comfortable with (the previous annotation attempts we’re lacking any annotations in almost all of the largest scaffolds, which didn’t seem right), Steven stumbled across GenSAS, a web/GUI-based genome annotation program, so we gave it a shot.

Read More

Genome Annotation - Olurida_v081 with MAKER and Tissue-specific Transcriptomes on Mox

  • 10 min read

I previously annotated our Olurida_v081 genome with MAKER using our “canonical” transcriptome, Olurida_transcriptome_v3.fasta as the EST evidence utilized by MAKER. A discussion on one of our Slack channels related to the lack of isoform annotation (I think it’s a private channel, sorry) prompted Katherine Silliman to suggest re-running the annotation using tissue-specific transcriptome assemblies that she has generated as EST evidence, instead of a singular transcriptome. Since I already had previous versions of the MAKER script that I’ve used for annotations, re-running was rather straightforward. While this was running, I used Stringtie on 20190625to produce a GTF that maps out potential isoforms, as I don’t believe MAKER will actually predict isoforms, since it didn’t do so the first time, nor has it with other annotations we’ve run on geoduck assemblies.

Read More

FastQC-MultiQC - Additional C.gigas WGBS Sequencing Data from Genewiz Received 20190501

  • ~1 min read

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.

Read More

Data Analysis - C.virginica MBD Sequencing Coverage

  • 2 min read

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.

Read More

Metagenomics Gene Prediction - P.generosa Water Samples Using MetaGeneMark on Mox to Compare pH Treatments

  • 2 min read

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.

Read More

Metagenome Assemblies - P.generosa Water Samples Trimmed HiSeqX Data Using Megahit on Mox to Compare pH Treatments

  • 2 min read

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:

Read More

RNA Isolation and Quantification - Crab Hemolypmh Using Quick-DNA-RNA Microprep Plus Kit

  • 1 min read

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:

Read More

Transcriptome Assembly - Geoduck Tissue-specific Assembly Larvae Day5 EPI99 with HiSeq and NovaSeq Data on Mox

  • 2 min read

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.

Read More