Yaamini’s Notebook: Gigas Broodstock DNA Extraction Part 7

Extraction plan for actual samples

Kaitlyn ran DNA samples from protocol test 3 and test 4 on the Bioanalyzer. You can see her results in this notebook post. Based on her findings, I need to use a TissueTearor at setting 1 for 10 seconds to lyse the samples before incubation. I will also remove RNA from the DNA samples. The official protocol can be found here.

I have 10 gonad samples per pH treatment (ambient or low). Because 20 samples is a lot to work with at once, I’ll do 2 days of DNA extractions with 10 samples each. I randomly selected which ambient and low pH samples to extract in each batch. The IDs correspond to names on histology photos in this folder. Below are diagrams for each histology block:

gigas1

gigas2

gigas3

gigas4

gigas5

Figures 1-5. Location of each tissue sample on histology blocks.

Batch 1: I’ll extract DNA from this batch tomorrow.

Low pH:

  • 6-T1
  • 9-T2
  • 2-T1
  • 5-T3
  • 4-T3

Ambient pH:

  • UK-07
  • 12-T6
  • UK-04
  • UK-03
  • UK-01

Batch 2: I’ll extract DNA from this batch on Tuesday.

Low pH:

  • 1-T3
  • 7-T2
  • 8-T2
  • 10-T3
  • 3-T1

Ambient pH:

  • UK-06
  • 11-T4
  • UK-05
  • UK-08
  • UK-02

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Kaitlyn’s notebook: Bioanalzyer results

I ran Yaamini’s samples on the bioanalyzer and got the results from the procedure tests for broodstock C. gigas DNA extraction from blocks.

 

Sam’s Notebook: Bedgraph – Olympia oyster transcriptome with Olurida_v081 genome assembly

0000-0002-2747-368X

I took the sorted BAM file from yesterday’s corrected RNAseq genome alignment and converted it to a bedgraph using BEDTools genomeCoverageBed tool.

Analysis took place on our HPC Mox node.

SBATCH script file:

  #!/bin/bash ## Job Name #SBATCH --job-name=20180926_oly_bedgraphs ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=5-00:00:00 ## Memory per node #SBATCH --mem=500G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --workdir=/gscratch/scrubbed/samwhite/20180926_oly_RNAseq_bedgraphs # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) date >> system_path.log echo "" >> system_path.log echo "System PATH for $SLURM_JOB_ID" >> system_path.log echo "" >> system_path.log printf "%0.s-" {1..10} >> system_path.log echo ${PATH} | tr : \\n >> system_path.log # Set sorted transcriptome assembly bam file oly_transcriptome_bam=/gscratch/scrubbed/samwhite/20180925_oly_RNAseq_genome_hisat2/20180925_Olurida_v081.sorted.bam # Set program paths bedtools=/gscratch/srlab/programs/bedtools-2.27.1/bin samtools=/gscratch/srlab/programs/samtools-1.9/samtools # Create bedgraph ## Reports depth at each position (-bg in bedgraph format) and report regions with zero coverage (-a). ## Screens for portions of reads coming from exons (-split). ## Add genome browser track line to header of bedgraph file. ${bedtools}/genomeCoverageBed \ -ibam ${oly_transcriptome_bam} \ -bga \ -split \ -trackline \ > 20180926_oly_RNAseq.bedgraph 

Sam’s Notebook: Transcriptome Alignment – Olympia oyster RNAseq reads aligned to genome with HISAT2

0000-0002-2747-368X

Yesterday’s attempt at producing a bedgraph was a failure and a prodcuct of a major brain fart. The worst part is that I was questioning what I was doing the entire time, but still went through with the process! Yeesh!

The problem was that I tried to take our Trinity-assembled transcriptome and somehow align that to our genome. This can’t work because each of those assemblies don’t know the coordinates used by the other. So, as was the case, you end up with a bedgraph that shows zero coverage for all genome contigs.

Anyway, here’s the correct procedure!

Used HISAT2 on our HPC Mox node to align our RNAseq reads to our Olurida_v081 genome assembly:

SBATCH script files:

PERFORM GENOME INDEXING & ALIGNMENT
20180925_oly_RNAseq_genome_hisat2.sh

SORT & INDEX ALIGNMENT OUTPUT
20180925_oly_RNAseq_genome_sort_bam.sh