Projects

Splice Site Identification - S.namaycush Liver Parasitized and Non-Parasitized SRA RNAseq Using Hisat2-Stingtie with Genome GCF_016432855.1

After previously downloading/trimming/QCing S.namaycush SRA liver RNAseq data on 20220706, Steven asked that I run through Hisat2 for splice site identification (GitHub Issue).

qPCR - Repeat of Mussel Gill Heat Stress cDNA with Ferritin Primers

My previous qPCR on these cDNA using ferritin primers (SRIDs: 1808, 1809) resulted in no amplification. This was a bit surprising and makes me suspect that I screwed up somewhere (not adding primer(s)??), so I decided to repeat the qPCR. I made fresh working primer stocks and used 1uL of cDNA for each reaction. All reactions were run in duplicate on our CFX Connect thermalcycler (BioRad) with SsoFast EVAgreen Master Mix (BioRad). See my previous post linked above for qPCR master mix calcs.

qPCR - Dorothys Mussel cDNA from 20220726

Ran qPCRs on Dorothy’s mussel gill cDNA from 20220726 using the following primers:

RNA Isolation and Quantification - Dorothy Mussel Gill Samples

Isolated RNA from a subset of Dorothy’s mussel gill samples:

BS-seq and SNP Analysis - Nextflow EpiDiverse Pipelines Trials and Tribulations

Alrighty, this notebook entry is going to have a lot to unpack, as the process to get these pipelines running and then deal with the actual data we wanted to run them with was quite involved. However, the TL;DR of this all is this:

SRA Data - S.namaycush SRA BioProject PRJNA674328 Download and QC

Per this GitHub Issue, which I accidentally forgot about for three weeks (!), Steven wanted me to download the lake trout (Salvelinus namaycush) RNAseq data from NCBI BioProject PRJNA674328 and run QC on the data.

Data Wrangling - Create Primary P.generosa Genome Annotation File

Steven asked me to create a canonical genome annotation file (GitHub Issue). I needed/wanted to create a file containing gene IDs, SwissProt (SP) IDs, gene names, gene descriptions, and gene ontology (GO) accessions. To do so, I utilized the NCBI BLAST and DIAMOND BLAST annotations generated by our GenSas P.generosa genome annotation. Per Steven’s suggestion, I used the best match (i.e. lowest e-value) for any given gene between the two files.

RNA Isolation - O.nerka Berdahl Brain Tissues

Steven asked that I isolate RNA from the remainder of the Berdahl brain tissue samples (GitHub issue).

Nextflow - Trials and Tribulations of Installing and Using NF-Core RNAseq

INSTALLATION

Data Wrangling - P.generosa Genomic Feature FastA Creation

Steven wanted me to generate FastA files (GitHub Issue) for Panopea generosa (Pacific geoduck) coding sequences (CDS), genes, and mRNAs. One of the primary needs, though, was to have an ID that could be used for downstream table joining/mapping. I ended up using a combination of GFFutils and bedtools getfasta. I took advantage of being able to create a custom name column in BED files to generate the desired FastA description line having IDs that could identify, and map, CDS, genes, and mRNAs across FastAs and GFFs.

Differential Gene Expression - P.generosa DGE Between Tissues Using Nextlow NF-Core RNAseq Pipeline on Mox

Steven asked that I obtain relative expression values for various geoduck tissues (GitHub Issue). So, I decided to use this as an opportunity to try to use a Nextflow pipeline. There’s an RNAseq pipeline, NF-Core RNAseq which I decided to use. The pipeline appears to be ridiculously thorough (e.g. trims, removes gDNA/rRNA contamination, allows for multiple aligners to be used, quantifies/visualizes feature assignments by reads, performs differential gene expression analysis and visualization), all in one package. Sounds great, but I did have some initial problems getting things up and running. Overall, getting things set up to actually run took longer than the actual pipeline run! Oh well, it’s a learning process, so that’s not totally unexpected.

Data Analysis - C.virginica BSseq Unmapped Reads Using MEGAN6

After performing DIAMOND BLASTx and DAA “meganization” on 20220302, the next step was to import the DAA files into MEGAN6 for analyzing the resulting taxonomic assignments of the Crassostrea virginica (Eastern oyster) unmapped BSseq reads that Steven generated.

Data Analysis - C.virginica RNAseq Zymo ZR4059 Analyzed by ZymoResearch

After realizing that the Crassostrea virginica (Eastern oyster) RNAseq data had relatively low alignment rates (see this notebook entry from 20220224 for a bit more background), I contacted ZymoResearch to see if they had any insight on what might be happening. I suspected rRNA contamination. ZymoResearch was kind enough to run the RNAseq data through their pipeline and provided us. This notebook entry provides a brief overview and thoughts on the report.

Taxonomic Assignment - C.virginica BSseq Unmapped Reads Using DIAMOND BLASTx and MEGAN6 on Mox

After mapping bisulfite sequencing (BSseq) data to the Crassostrea virginica (Eastern oyster) genome, Steven noticed that there were a large number of unmapped reads. He asked that I attempt to taxonomically claissify the unmapped reads (GitHub Issue), with the idea that maybe these reads could provide additional data on an associated microbiome (GitHub Discussion).

Data Wrangling - P.generosa Genome GFF Conversion to GTF Using gffread

Steven asked in this GitHub Issue to convert our Panopea generosa (Pacific geoduck) genomic GFF to a GTF for use in the 10x Genomics Cell Ranger software. This conversion was performed using GffRead in a Jupyter Notebook.

Transcript Identification and Alignments - C.virginica RNAseq with NCBI Genome GCF_002022765.2 Using Hisat2 and Stringtie on Mox

After an additional round of trimming yesterday, I needed to identify alternative transcripts in the Crassostrea virginica (Eastern oyster) gonad RNAseq data we have. I previously used HISAT2 to index the NCBI Crassostrea virginica (Eastern oyster) genome and identify exon/splice sites on 20210720. Then, I used this genome index to run StringTie on Mox in order to map sequencing reads to the genome/alternative isoforms.

Trimming - Additional 20bp from C.virginica Gonad RNAseq with fastp on Mox

When I previously aligned trimmed RNAseq reads to the NCBI C.virginica genome (GCF_002022765.2) on 20210726, I specifically noted that alignment rates were consistently lower for males than females. However, I let that discrepancy distract me from a the larger issue: low alignment rates. Period! This should have thrown some red flags and it eventually did after Steven asked about overall alignment rate for an alignment of this data that I performed on 20220131 in preparation for genome-guided transcriptome assembly. The overall alignment rate (in which I actually used the trimmed reads from 20210714) was ~67.6%. Realizing this was a on the low side of what one would expect, it prompted me to look into things more and I came across a few things which led me to make the decision to redo the trimming:

Data Wrangling - C.virginica lncRNA Extractions from NCBI GCF_002022765.2 Using GffRead

Continuing to work on our Crassostrea virginica (Eastern oyster) project examining the effects of OA on female and male gonads (GitHub repo), Steven tasked me with parsing out long, non-coding RNAs (GitHub Issue). To do so, I relied on the NCBI genome and associated files/annotations. I used GffRead, GFFutils, and samtools. The process was documented in the followng Jupyter Notebook:

Transcriptome Assembly - Genome-guided C.virginica Adult Gonad OA RNAseq Using Trinity on Mox

As part of this project, Steven’s asked that I identify long, non-coding RNAs (lncRNAs) (GitHub Issue) in the Crassostrea gigas (Pacific oyster) adult OA gonad RNAseq data we have. The initial step for this is to assemble transcriptome. I generated the necessary BAM alignment on 20220131. Next was to actually get the transcriptome assembled. I followed the Trinity genome-guided procedure.

RNAseq Alignment - C.virginica Adult OA Gonad Data to GCF_002022765.2 Genome Using HISAT2 on Mox

As part of this project, Steven’s asked that I identify long, non-coding RNAs (lncRNAs) (GitHub Issue) in the Crassostrea virginica (Eastern oyster) adult OA gonad RNAseq data we have. The initial step for this is to assemble transcriptome. Since there is a published genome (NCBI RefSeq GCF_002022765.2C_virginica-3.0)](https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/002/022/765/GCF_002022765.2_C_virginica-3.0/) for [_Crassostrea virginica (Eastern oyster), I will perform a genome-guided assembly using Trinity. That process requires a sorted BAM file as input. In order to generate that file, I used HISAT2. I’ve already generated the necessary HISAT2 genome index files (as of 20210720), which also identified/incorporated splice sites and exons, which the HISAT2 alignment process requires to run.

Data Wrangling - C.virginica Gonad RNAseq Transcript Counts Per Gene Per Sample Using Ballgown

As we continue to work on the analysis of impacts of OA on Crassostrea virginica (Eastern oyster) gonads via DNA methylation and RNAseq (GitHub repo), we decided to compare the number of transcripts expressed per gene per sample (GitHub Issue). As it turns out, it was quite the challenge. Ultimately, I wasn’t able to solve it myself, and turned to StackOverflow for a solution. I should’ve just done this at the beginning, as I got a response (and solution) less than five minutes after posting! Regardless, the data wrangling progress (struggle?) was documented in the following GitHub Discussion:

RNA Isolation - O.nerka Berdahl Brain Tissues

The previous round of RNA isolation on 20211222 had poor yields for the brain tissue. As such, I needed to attempt to obtain RNA from some additional brain tissues.

RNA Isolation - M.trossulus Gill

As part of a mussel project that Matt George has with the Pacific States Marine Fisheries Commission (PSMFC), I’m helping by continuing isolating RNA from a relatively large number of samples. The samples are listed/described in this GitHub Issue. Today, I isolated RNA from the following samples (the “F” indicates “foot”, “PG” indicates “phenol gland”, and “G” indicates “gill” tissues):

RNA Isolation - M.trossulus Gill and Phenol Gland

As part of a mussel project that Matt George has with the Pacific States Marine Fisheries Commission (PSMFC), I’m helping by continuing isolating RNA from a relatively large number of samples. The samples are listed/described in this GitHub Issue. Today, I isolated RNA from the following samples (the “F” indicates “foot”, “PG” indicates “phenol gland”, and “G” indicates “gill” tissues):

RNA Isolation - M.trossulus Gill and Phenol Gland

As part of a mussel project that Matt George has with the Pacific States Marine Fisheries Commission (PSMFC), I’m helping by continuing isolating RNA from a relatively large number of samples. The samples are listed/described in this GitHub Issue. Today, I isolated RNA from the following samples (the “F” indicates “foot”, “PG” indicates “phenol gland”, and “G” indicates “gill” tissues):

RNA Isolation - M.trossulus Gill and Phenol Gland

As part of a mussel project that Matt George has with the Pacific States Marine Fisheries Commission (PSMFC), I’m helping by continuing isolating RNA from a relatively large number of samples. The samples are listed/described in this GitHub Issue. Today, I isolated RNA from the following samples (the “F” indicates “foot”, “PG” indicates “phenol gland”, and “G” indicates “gill” tissues):

RNA Isolation - M.trossulus Phenol Gland and Gill

As part of a mussel project that Matt George has with the Pacific States Marine Fisheries Commission (PSMFC), I’m helping by continuing isolating RNA from a relatively large number of samples. The samples are listed/described in this GitHub Issue. Today, I isolated RNA from the following samples (the “F” indicates “foot”, “PG” indicates “phenol gland”, and “G” indicates “gill” tissues):

RNA Isolation - M.trossulus Foot and Phenol Gland

As part of a mussel project that Matt George has with the Pacific States Marine Fisheries Commission (PSMFC), I’m helping by isolating RNA from a relatively large number of samples. The samples are listed/described in this GitHub Issue. Today, I isolated RNA from the following samples (the “F” indicates “foot” and the “PG” indicates “phenol gland” tissues):

RNA Isolation - O.nerka Berdahl Brain Tissues

The previous round of RNA isolation (on 20211220) had poor yields for the brain tissue. As such, I needed to attempt to obtain RNA from some additional brain tissues.

RNA Isolation - O.nerka Berdahl Tissues

Finally got around to tackling this GitHub issue regarding isolating RNA from some Oncorhynchus nerka (sockeye salmon) tissues we have from Andrew Berdahl’s lab (a UW SAFS professor) to use for RNAseq and/or qPCR. We have blood, brain, gonad, and liver samples from individual salmon from two different groups: territorial and social individuals. We’ve decided to isolate RNA from brain, gonads, and liver from two individuals within each group. All samples are preserved in RNAlater and stored @ -80^oC.

Data Wrangling - C.virginica NCBI GCF_002022765.2 GFF to Gene BED File

When working to identify differentially expressed transcripts (DETs) and genes (DEGs) for our Crassostrea virginica (Eastern oyster) RNAseq/DNA methylation comparison of changes across sex and ocean acidification conditions (https://github.com/epigeneticstoocean/2018_L18-adult-methylation), I realized that the DEG tables I was generating had excessive gene counts due to the fact that the analysis (and, in turn, the genome coordinates), were tied to transcripts. Thus, genes were counted multiple times due to the existence of multiple transcripts for a given gene, and the analysis didn’t list gene coordinate data - only transcript coordinates.

Differential Transcript Expression - C.virginica Gonad RNAseq Using Ballgown

In preparation for differential transcript analysis, I previously ran our RNAseq data through StringTie on 20210726 to identify and quantify transcripts. Identification of differentially expressed transcripts (DETs) and genes (DEGs) will be performed using ballgown. This notebook entry will be different than most others, as this notebook entry will simply serve as a “landing page” to access/review the analysis; as the analysis will evolve over time and won’t exist as a single computing job with a definitive endpoint.

Transcript Identification and Quantification - C.virginia RNAseq With NCBI Genome GCF_002022765.2 Using StringTie on Mox

After having run HISAT2 to index and identify exons and splice sites in the NCBI Crassostrea virginica (Eastern oyster) genome (GCF_002022765.2) on 20210720, the next step was to identify and quantify transcripts from the RNAseq data using StringTie.

Genome Annotations - Splice Site and Exon Extractions for C.virginica GCF_002022765.2 Genome Using Hisat2 on Mox

Previously performed quality trimming on the Crassostrea virginica (Eastern oyster) gonad/sperm RNAseq data on 20210714. Next, I needed to identify exons and splice sites, as well as generate a genome index using HISAT2 to be used with StringTie downstream to identify potential alternative transcripts. This utilized the following NCBI genome files:

Trimming - C.virginica Gonad RNAseq with FastP on Mox

Needed to trim the Crassostrea virginica (Eastern oyster) gonad RNAseq data we received on 20210528.

FastQC-MultiQC - Yaamini’s C.virginica RNAseq and WGBS from ZymoResearch on Mox

Finally got around to running FastQC on Yaamini’s RNAseq and WGBS sequencing data recieved on 20210528.

Data Wrangling - S.salar Gene Annotations from NCBI RefSeq GCF_000233375.1_ICSASG_v2_genomic.gff for Shelly

Shelly posted a GitHub Issue asking if I could create a file of S.salar genes with their UniProt annotations (e.g. gene name, UniProt accession, GO terms).

RepeatMasker - C.gigas Rosling NCBI Genome GCA_902806645.1 on Mox

Decided to tackle this GitHub Issue about creating a transposable elements IGV track with the new Roslin C.gigas genome, since it had been sitting for a while and I have code sitting around that’s ready to roll for this type of thing.

Singularity - RStudio Server Container on Mox

Aidan recently needed to use R on a machine with more memory. Additionally, it would be ideal if he could use RStudio. So, I managed to figure out how to set up a Singularity container running rocker/rstudio.

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

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).

Genome Annotation - P.generosa v1.0 Assembly Using DIAMOND BLASTx for BlobToolKit on Mox

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 DIAMOND BLASTx according to the BlobToolKit “Getting Started” guide on Mox.

Genome Annotation - P.generosa v1.0 Assembly Using BLASTn for BlobToolKit on Mox

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 BLASTn according to the BlobToolKit “Getting Started” guide on Mox.

Trimming P.generosa 10x Genomics HiC FastQs with fastp on Mox

Steven asked me to try running Blob Tool Kit to identify potential contaminating sequence in our Panopea generosa (Pacific geoduck) genome assembly (v1.0). In preparation for running Blob Tool Kit, I needed to trim the 10x Genomics FastQ data used by Phase Genomics. Files were trimmed using fastp on Mox.

Transcriptome Annotation - Trinotate on C.bairdi Transcriptome v4.0 on Mox

Continued 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 Trinotate on Mox. This will provide a thorough annotation, including genoe ontology (GO) term assignments to each contig.

Transcriptome Annotation - DIAMOND BLASTx on C.bairdi Transcriptome v4.0 on Mox

Continued 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 DIAMOND BLASTx on Mox. This will be used as a component of Trinotate annotation downstream.

Transcriptome Annotation - Trinotate Hematodinium v1.7 on Mox

To continue annotation of our Hematodinium v1.7 transcriptome assembly, I wanted to run Trinotate.

Transcriptome Annotation - Trinotate Hematodinium v1.6 on Mox

To continue annotation of our Hematodinium v1.6 transcriptome assembly, I wanted to run Trinotate.

Transcriptome Assembly - Hematodinium Transcriptomes v1.6 and v1.7 with Trinity on Mox

I’d previously assembled hemat_transcriptome_v1.0.fasta on 20200122, hemat_transcriptome_v1.5.fasta on 20200408, extracted hemat_transcriptome_v2.1.fasta from an existing FastA on 20200605, as well as extracted hemat_transcriptome_v3.1.fasta on 20200605.

Data Wrangling - Gene ID Extraction from P.generosa Genome GFF Using Methylation Machinery Gene IDs

Per this GitHub issue, Steven provided a list of methylation-related gene names and wanted to extract the corresponding Panopea generosa ([Pacific geoduck (Panopea generosa)](http://en.wikipedia.org/wiki/Geoduck)) gene ID from our P.generosa genome, along with corresponding BLAST e-values.

Data Wrangling - Gene ID Extraction from P.generosa Genome GFF Using Methylation Machinery List

Per this GitHub Issue Steven asked that I take a list of gene names associated with DNA methylation and see if I could extract a list of Panopea generosa (Panopea generosa) gene IDs and corresponding BLAST e-values for each from our P.generosa genome annotation (see Genomic Resources wiki for more info).

Data Received - Anthopleura elegantissima - aggregating anenome - NanoPore Genome Sequence from Jay Dimond

Jay asked me to help get his A.elegantissima (aggregating anenome) NanoPore gDNA sequencing data submitted to NCBI Sequencing Read Archive (SRA). He sent a hard drive (HDD) with all the NanoPore sequencing Fast5 files. The HDD was received on 2/2/2021. Here’re are details provided in the reamde file in the Ae_ONT directory.

Samples Submitted - M.magister MBD-BSseq Libraries to Univ. of Oregon GC3F

Submitted the M.magister MBD-BSseq libraries created 20201124 using the 4nM aliquots created for the MiSeq test run on 20201202 to the Univ. of Oregon GC3F sequencing core.

FastQC-MultiQC - M.magister MBD-BSseq Pool Test MiSeq Run on Mox

Earlier today we received the M.magister (C.magister; Dungeness crab) MiSeq data from Mac.

Data Received - M.magister MBD-BSseq Pool Test MiSeq Run

After creating _M.magister (C.magister; Dungeness crab) MBD-BSseq libraries (on 20201124), I gave the pooled set of samples to Mac for a test sequencing run on the MiSeq on 20201202.

Alignment - C.gigas RNAseq to GCF_000297895.1_oyster_v9 Genome Using STAR on Mox

Mac was getting some weird results when mapping some single cell RNAseq data to the C.gigas mitochondrial (mt) genome that she had, so she asked for some help mapping other C.gigas RNAseq data (GitHub Issue) to the C.gigas mt genome to see if someone else would get similar results.

Trimming - Haws Lab C.gigas Ploidy pH WGBS 10bp 5 and 3 Prime Ends Using fastp and MultiQC on Mox

Making the assumption that the 24 C.gigas ploidy pH WGBS data we receved 20201205 will be analyzed using Bismark, I decided to go ahead and trim the files according to Bismark guidelines for libraries made with the ZymoResearch Pico MethylSeq Kit.

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

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.

Trimming - Ronits C.gigas Ploidy WGBS 10bp 5 and 3 Prime Ends Using fastp and MultiQC on Mox

Steven asked me to trim (GitHub Issue) Ronit’s WGBS sequencing data we received on 20201110, according to Bismark guidelines for libraries made with the ZymoResearch Pico MethylSeq Kit.

Library Quantification - M.magister MBD BSseq Libraries with Qubit

After reviewing the Bionalyzer assays for the MBD BSseq libraries Mac indicated she’d like to have the libraries quantified using the Qubit.

Trimming - Ronits C.gigas Ploidy WGBS Using fastp and MultiQC on Mox

Steven asked me to trim (GitHub Issue) Ronit’s WGBS sequencing data we received on 20201110, according to Bismark guidelines for libraries made with the ZymoResearch Pico MethylSeq Kit.

Bioanalyzer - M.magister MBD BSseq Libraries

MBD BSseq library construction was completed yesterday (20201124). Next, I needed to evaluate the libraries using the Roberts Lab Bioanalyzer 2100 (Agilent) to assess library sizes, yields, and qualities (i.e. primer dimers).

MBD BSseq Library Prep - M.magister MBD-selected DNA Using Pico Methyl-Seq Kit

After finishing the final set of eight MBD selections on 20201103, I’m finally ready to make the BSseq libraries using the Pico Methyl-Seq Library Prep Kit (ZymoResearch) (PDF). I followed the manufacturer’s protocols with the following notes/changes (organized by each section in the protocol):

RNA Isolation and Quantification - P.generosa Hemocytes from Shelly

Shelly asked me to isolate RNA from some P.generosa hemocytes (GitHub Issue) that she had.

FastQC-MultiQc - C.gigas Ploidy WGBS Raw Sequence Data from Ronits Project on Mox

Earlier today, we received the C.gigas ploidy WGBS data that we submitted to ZymoResearch on 20200820.

Transcriptome Assessment - Crustacean Transcripome Completeness Evaluation Using BUSCO on Mox

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.

Data Wrangling - MultiQC on S.salar RNAseq from fastp and HISAT2 on Mox

In Shelly’s GitHub Issue for this S.salar project, she also requested a MultiQC report for the trimming (completed on 20201029) and the genome alignments (completed on 20201103).

RNAseq Alignments - S.salar HISAT2 BAMs to GCF_000233375.1_ICSASG_v2_genomic.gtf Transcriptome Using StringTie on Mox

This is a continuation of addressing Shelly Trigg’s (regarding some Salmo salar RNAseq data) request (GitHub Issue) to trim (completed 20201029), perform genome alignment (completed on 20201103), and transcriptome alignment.

RNAseq Alignments - Trimmed S.salar RNAseq to GCF_000233375.1_ICSASG_v2_genomic.fa Using Hisat2 on Mox

This is a continuation of addressing Shelly Trigg’s (regarding some Salmo salar RNAseq data) request (GitHub Issue) to trim (completed 20201029), perform genome alignment, and transcriptome alignment.

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

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).

MBD Selection - M.magister Sheared Gill gDNA 8 of 24 Samples Set 2 of 3

Click here for notebook on the first 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).

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

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.

MBD Selection - M.magister Sheared Gill gDNA 8 of 24 Samples Set 1 of 3

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).

DNA Shearing - M.magister gDNA Additional Shearing CH05-01_21 CH07-11 and Bioanalyzer

After shearing all of the M.magister gill gDNA on 20201026, there were still three samples that still had average fragment lengths that were a bit longer than desired (~750bp, but want ~250 - 550bp):

DNA Shearing - M.magister gDNA Shearing All Samples and Bioanalyzer

I previously ran some shearing tests on 20201022 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 was 70 cycles (30s ON, 30s OFF; low intensity), sonicating for 35 cycles, followed by successive rounds of 5 cycles each.

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

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.

DNA Shearing - M.magister gDNA Shear Testing and Bioanalyzer

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 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

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.

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

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.

NanoPore Reads Extractions - C.bairdi Taxonomic Reads Extractions with MEGAN6 on 201002558-2729-Q7 and 6129-403-26-Q7

After completing the taxonomic comparisons of 201002558-2729-Q7 and 6129-403-26-Q7 on 20201002, I decided to extract reads assigned to the following taxa for further exploration (primarily to identify contigs/scaffolds in our cbai_genome_v1.0.fasta (19MB).

Comparison - C.bairdi 20102558-2729 vs. 6129-403-26 NanoPore Taxonomic Assignments Using MEGAN6

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.

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

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.

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

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.

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

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.

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

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.

Assembly Assessment - BUSCO C.bairdi Genome v1.01 on Mox

After creating a subset of the cbai_genome_v1.0 of contigs >100bp yesterday (subset named cbai_genome_v1.01), I wanted to generate BUSCO scores for cbai_genome_v1.01. This is primarily just to keep info consistent on our Genomic Resources wiki, as I don’t expect these scores to differ at all from the cbai_genome_v1.0 BUSCO scores.

Data Wrangling - Subsetting cbai_genome_v1.0 Assembly with faidx

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.

Assembly Assessment - BUSCO C.bairdi Genome v1.0 on Mox

After using Flye to perform a de novo assembly of our Q7 filtered NanoPore sequencing data on 20200917, I decided to check the “completeness” of the assembly using BUSCO on Mox.

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

I previously converting our C.bairdi NanoPre sequencing data from the raw Fast5 format to FastQ format for our three sets of data:

Taxonomic Assignments - C.bairdi NanoPore Reads Using DIAMOND BLASTx on Mox and MEGAN6 daa2rma on swoose

Earlier today I quality filtered (>=Q7) our C.baird NanoPore reads. One of the things I’d like to do now is to attempt to filter reads taxonomically, since the NanoPore data came from both an uninfected crab and Hematodinium-infected crab.

qPCR - Geoduck Normalizing Gene Primers 28s-v4 and EF1a-v1 Tests

On Monday (20200914), I checked a set of 28s and EF1a primer sets and determined that 28s-v4 and EF1a-v1 were probably the best of the bunch, although they all looked great. So, I needed to test these out on some individual cDNA samples to see if they might be useful as normalizing genes - should have consistent Cq values across all samples/treatments.

qPCR - Geoduck Normalizing Gene Primer Checks

Shelly ordered some new primers (designed by Sam Gurr) (GitHub Issue) to potentially use as normalizing genes for her geoduck reproduction gene expression project and asked that I test them out.

Data Wrangling - Visualization of C.bairdi NanoPore Sequencing Using NanoPlot on Mox

I previously converting our C.bairdi NanoPre sequencing data from the raw Fast5 format to FastQ format for our three sets of data:

Data Wrangling - NanoPore Fast5 Conversion to FastQ of C.bairdi 6129_403_26 on Mox with GPU Node

Time to start working with the NanoPore data that I generated back in March (???!!!). In order to proceed, I first need to convert the raw Fast5 files to FastQ. To do so, I’ll use the NanoPore program guppy.

Data Wrangling - NanoPore Fast5 Conversion to FastQ of C.bairdi 20102558-2729 Run-02 on Mox with GPU Node

Continuing to work with the NanoPore data that I generated back in January(???!!!). In order to proceed, I first need to convert the raw Fast5 files to FastQ. To do so, I’ll use the NanoPore program guppy. I converted the first run from this flowcell earlier today.

Data Wrangling - NanoPore Fast5 Conversion to FastQ of C.bairdi 20102558-2729 Run-01 on Mox with GPU Node

Time to start working with the NanoPore data that I generated back in January(???!!!). In order to proceed, I first need to convert the raw Fast5 files to FastQ. To do so, I’ll use the NanoPore program guppy.

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

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.

Transcriptome Annotation - Trinotate Hematodinium v3.1 on Mox

To continue annotation of our Hematodinium v3.1 transcriptome assembly, I wanted to run Trinotate.

Transcriptome Annotation - Trinotate Hematodinium v2.1 on Mox

To continue annotation of our Hematodinium v1.6 transcriptome assembly, I wanted to run Trinotate.

qPCR - P.generosa RPL5 and TIF3s6b v2 and v3 Normalizing Gene Assessment

After testing out the RPL5 and TIF3s6b v2 and v3 primers yesterday on pooled cDNA, we determined the primers looked good, so will go forward testing them on a set of P.generosa hemolymph cDNA made by Kaitlyn on 20200212. This will evaluate whether or not these can be utilized as normalizing genes for subsequent gene expression analyses.

Sample Submitted - C.gigas Diploid-Triploid pH Treatments Ctenidia to ZymoResearch for WGBS

Submitted 1.5ug of the 24 C.gigas ctenidia ctenidia gDNA isolated last week (20200821) to ZymoResearch for whole genome bisulfite sequencing (WGBS) to compare differences in diploid/triploids and responses to elevated pH:

qPCR - P.generosa RPL5-v2-v3 and TIF3s6b-v2-v3 Primer Tests

Shelly ordered some new primers as potential normalizing genes and asked me to test them out (GitHub Issue).

DNA Isolation and Quantification - C.gigas High-Low pH Triploid and Diploid Ctenidia

Isolated DNA from 24 of the Crassostrea gigas high/low pH triploid/diploid ctenidia samples that we received yesterday from the Haws Lab. Samples selected by Steven.

Samples Submitted - Ronits C.gigas Diploid and Triploid Ctenidia to ZymoResearch for WGBS

Submitted 1.75ug of gDNA from 10 Crassostrea gigas ctenidia samples from Ronit’s dessication/temp/ploidy experiment to ZymoResearch for whole genome bisulfite sequencing (BSseq). They will sequence to ~30x coverage, using 150bp PE reads.

Assembly Stats - C.bairdi Transcriptomes v2.1 and v3.1 Trinity Stats on Mox

Realized that transcriptomes v2.1 and v3.1 (extracted from BLASTx-annotated FastAs from 20200605) didn’t have any associated stats.

Trimming-FastQC-MultiQC - Robertos C.gigas WGBS FastQ Data with fastp FastQC and MultiQC on Mox

Steven asked me to trim Roberto’s C.gigas whole genome bisulfite sequencing (WGBS) reads (GitHub Issue) “following his methods”. The only thing specified is trimming Illumina adaptors and then trimming 10bp from the 5’ end of reads. No mention of which software was used.

TransDecoder - Hematodinium Transcriptomes v1.6, v1.7, v2.1 and v3.1 on Mox

To continue annotation of our Hematodinium v1.6, v1.7, v2.1 & v3.1 transcriptome assemblies, I needed to run TransDecoder before performing the more thorough annotation with Trinotate.

Assembly Stats - cbaiodinium Transcriptomes v2.1 and v3.1 Trinity Stats on Mox

Working on dealing with our various cbaiodinium sp. transcriptomes and realized that transcriptomes v2.1 and v3.1 (extracted from BLASTx-annotated FastAs from 20200605) didn’t have any associated stats.

Transcriptome Assessment - BUSCO Metazoa on Hematodinium v1.6 v1.7 v2.1 and v3.1 on Mox

Needed to assess the Hematodinium sp. transcriptomes that I’ve assembled to determine their “completeness” using BUSCO.

Transcriptome Annotation - Hematodinium Transcriptomes v1.6 v1.7 v2.1 v3.1 with DIAMOND BLASTx on Mox

Needed to annotate the Hematodinium sp. transcriptomes that I’ve assembled using DIAMOND BLASTx. This will also be used for additional downstream annotation (TransDecoder, Trinotate):

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 Comparison - C.bairdi Transcriptomes Evaluations with DETONATE on Mox

Attempting to get some other metrics regarding our various C.bairdi transcriptome assemblies other than BUSCO scores, I decided to try running DETONATE, as this is a recommended tool by Trinity.

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

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.

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.

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.

NanoPore Sequencing - C.bairdi gDNA 6129_403_26

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.

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:

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).

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.

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.

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.

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.

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.

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:

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.

DNA Isolation and Quantification - C.bairdi Hemolymph Pellets in RNAlater

Isolated DNA from the following 23 samples:

Lab Maintenance - Cluster UPS Battery Replacement

Replaced the batteries on one of the APC uninterruptable power supplies (UPS) on our local server cabinet.

NanoPore Sequencing - C.bairdi gDNA Sample 20102558-2729

I performed the initial Lambda sequencing test on 20200107 and everything went smoothly, so I’m ready to give the NanoPore (ONT) MinION a run with an actual sample!

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.

NanoPore Sequencing - Initial NanoPore MinION Lambda Sequencing Test

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.

UPDATE 20191125

Miscellaneous

INSTALLATION