Yaamini’s Notebook: DML Analysis Part 12

Running bismark on Mox

Today I started my bismark run on Mox! Here’s how I did it:

Create a script

Since I’m running bismark with revised parameters, I’m putting these “clean” analyses in my class repository. In my class repository, I created a shell script to run bismark based on Sam’s script. Because I’m working in a script, I was able to specify variable paths for bismark, samtools, my genome folder, and my trimmed files.

Set up Mox

I navigated to the /gscratch/scrubbed/ directory and created a subdirectory called data. Within this subdirectory, I wanted to house my bisulfite genome and trimmed files. Steven and Sam said I could transfer these files to Mox in this issue. To do this, I initiated rsync transfers from Mox so I wouldn’t have to deal with two-factor authentication.

 rsync --archive --progress --verbose yaamini@172.25.149.226:/Users/yaamini/Documents/project-virginica-oa/analyses/2018-04-27-Bismark/2018-04-27-Bismark-Inputs . #Transfer my bisulfite genome to Mox  
 rsync --archive --progress --verbose yaamini@172.25.149.226:/Volumes/web/Athaliana/20180411_trimgalore_10bp_Cvirginica_MBD/zr2096_*R1*.fq.gz . #Transfer analysis files  

When I used ls -F to look at my directory contents, I realized I only transferred half of the trimmed files. I used the code below to transfer the rest.

 rsync --archive --progress --verbose yaamini@172.25.149.226:/Volumes/web/Athaliana/20180411_trimgalore_10bp_Cvirginica_MBD/zr2096_*_val_2.fq.gz . #Transfered rest of files  
 rsync --archive --progress --verbose yaamini@172.25.149.226:/Volumes/web/Athaliana/20180411_trimgalore_10bp_Cvirginica_MBD/checksums.md5 . #Transfer checksums  
 md5sum -c checksums.md5 #Check checksums. All passed! ```` Based on [Steven's feedback](https://github.com/RobertsLab/resources/issues/431#issuecomment-430770659), I transferred my shell script to my user directory.  

rsync –archive –progress –verbose yaamini@172.25.149.226://Users/yaamini/Documents/yaamini-virginica/scripts/2018-10-12-Bismark-Revised-Parameters.sh . #Transfer script to USER directory, not scrubbed directory

  ### Start Mox run Now that I had everything on Mox, I was ready to start running the job!  

sbatch -p srlab -A srlab 2018-10-12-Bismark-Revised-Parameters.sh #Submitted batch job 381386

  Then [that failed](https://github.com/RobertsLab/resources/issues/438#issuecomment-430776330)...because I didn't properly specify my SBATCH path. My output directory is within my data directory, so I specified that in my script on genefish, then moved the fixed script to Mox.  

sbatch -p srlab -A srlab 2018-10-12-Bismark-Revised-Parameters.sh #Submitted batch job 381403 ““`

Now I’m running! The more I think about it, the more I don’t like analysis output in my data directory. Once my job ends, I’ll move the output directory to an analyses subdirectory.

Fingers crossed this all works well.

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Sam’s Notebook: Reverse Transcription – Ronit’s C.gigas DNased ctenidia RNA

0000-0002-2747-368X

Proceeded with reverse transcription of Ronit’s DNased ctenidia RNA (from 20181016).

Reverse transcription was performed using 100ng of each sample with M-MMLV Reverse Transcriptase from Promega.

Briefly, 100ng of DNased RNA was combined with oligo dT primers and brought up to a final volume of 15uL. Tubes were incubated for 5mins at 70oC in a PTC-200 thermal cycler (MJ Research), using a heated lid. Samples were immediately placed on ice.

A master mix of buffer, dNTPs, water, and M-MMLV reverse transcriptase was made, 10uL of the master mix was added to each sample, and mixed via finger flicking. Samples were incubated for 1hr at 42oC in a PTC-200 thermal cycler (MJ Research), using a heated lid, followed by a 5min incubation at 65oC.

Samples were stored on ice for use later this afternoon by Ronit.

Samples will be stored in Ronit’s -20oC box.

Reverse transcription calcs (Google Sheet):

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Sam’s Notebook: qPCR – Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with elongation factor primers

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After I figured out the appropriate DNA and primers to use to detect gDNA in Crassostrea gigas samples, I checked Ronit’s DNased ctenidia RNA (from 20181016) for residual gDNA.

Elongation factor primers:

  • EF1_qPCR_5′ (SRID 309)
  • EF1_qPCR_3′ (SRID 310)

BB16 from 20090519 was used as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):

bowtie Phred64

sr320@mox1 ~]$ cat /gscratch/srlab/sr320/jobs/1017_0800.sh 
#!/bin/bash
## Job Name
#SBATCH --job-name=bow64
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes (We only get 1, so this is fixed)
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=5-100:00:00
## Memory per node
#SBATCH --mem=100G
#SBATCH --mail-type=ALL
#SBATCH --mail-user=sr320@uw.edu
## Specify the working directory for this job
#SBATCH --workdir=/gscratch/srlab/sr320/analyses/1017

source /gscratch/srlab/programs/scripts/paths.sh


for fastq in /gscratch/scrubbed/sr320/Phred64_fqs/*1.fq.gz; do
  read1=$(echo "$fastq")
  read1_array+=("$read1")
done

for fastq in /gscratch/scrubbed/sr320/Phred64_fqs/*2.fq.gz; do
  read2=$(echo "$fastq")
  read2_array+=("$read2")
done

for pair in "${!read1_array[@]}"; do
  i=${read1_array[$pair]}
  j=${read2_array[$pair]}
  filename="${i##*/}"
  no_ext="${filename%%.*}"
  /gscratch/srlab/programs/bowtie2-2.3.4.1-linux-x86_64/bowtie2 \
  -x /gscratch/srlab/sr320/data/Chinook_genome_scaffold_masked/chinook_genome_masked \
  -1 "$i" \
  -2 "$j" \
  -X 2000 --sensitive  --no-mixed --phred64 --no-discordant --no-unal \
  -S /gscratch/scrubbed/sr320/cw/1017/"$no_ext"_bowtie2.sam \
  -q \
  -p 28
done

[

#sbatch

Sam’s Notebook: Samples Received – Crassostrea virginica (Eastern oyster) tissue from Lotterhos Lab (Northeastern University)

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20181017_samples_recd.jpg

Sample sheet (Google Sheet):

Samples were stored in -80oC:

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Sam’s Notebook: qPCR – C.gigas primer and gDNA tests with 18s and EF1 primers

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The [qPCR I ran earlier today to check for residual gDNA in Ronit’s DNased RNA] turned out terribly, due to a combination of bad primers and, possibly, bad gDNA.

I tracked down some different primers for testing:

  • Cg_18s_1644_F (SRID 1168)
  • Cg_18s_1750_R (SRID 1169)
  • EF1_qPCR_5′ (SRID 309)
  • EF1_qPCR_3′ (SRID 310)

In addition to BB15 from 20090519, I decided to test out BB16 from 20090519 as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):

Sam’s Notebook: qPCR – Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with 18s primers

0000-0002-2747-368X

After DNasing Ronit’s RNA earlier today, I needed to check for any residual gDNA.

Identified some old, old C.gigas 18s primers that should amplify gDNA:

  • gigas18s_fw (SRID 157)
  • gigas18s_rv (SRID 156)

Used some old C.gigas gDNA (BB15 from 20090519) as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):