Category Archives: Miscellaneous

TrimGalore/FastQC/MultiQC – 14bp Trim C.virginica MBD BS-seq FASTQ data

Yesterday, I ran TrimGalore/FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch with the default settings (i.e. “auto-trim”). There was still some variability in the first ~15bp of the reads and Steven wanted to see how a hard trim would change things.

I ran TrimGalore (using the built-in FastQC option), with a hard trim of the first 14bp of each read and followed up with MultiQC for a summary of the FastQC reports.

TrimGalore job script:

Standard error was redirected on the command line to this file:

MD5 checksums were generated on the resulting trimmed FASTQ files:

All data was copied to my folder on Owl.

Checksums for FASTQ files were verified post-data transfer (data not shown).

Results:

Output folder:

FastQC output folder:

MultiQC output folder:

MultiQC HTML report:

OK, this trimming definitely took care of the variability seen in the first ~15bp of all the reads.

However, I noticed that the last 2bp of each of the Read 1 seqs all have some wonky stuff going on. I’m guessing I should probably trim that stuff off, too…

TrimGalore/FastQC/MultiQC – Auto-trim C.virginica MBD BS-seq FASTQ data

Yesterday, I ran FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch. Steven wanted to trim it and see how things turned out.

I ran TrimGalore (using the built-in FastQC option) and followed up with MultiQC for a summary of the FastQC reports.

TrimGalore job script:

Standard error was redirected on the command line to this file:

MD5 checksums were generated on the resulting trimmed FASTQ files:

All data was copied to my folder on Owl.

Checksums for FASTQ files were verified post-data transfer.

Results:

Output folder:

FastQC output folder:

MultiQC output folder:

MultiQC HTML report:

Overall, the auto-trim didn’t alter things too much. Specifically, Steven is concerned about the variability in the first 15bp (seen in the Per Base Sequence Content section of the MultiQC output). It was reduced, but not greatly. Will perform an independent run of TrimGalore and employ a hard trim of the first 14bp of each read and see how that looks.

FastQC/MultiQC – C. virginica MBD BS-seq Data

Per Steven’s GitHub Issues request, I ran FastQC on the Eastern oyster MBD bisulfite sequencing data we recently got back from ZymoResearch.

Ran FastQC locally with the following script: 20180409_fastqc_Cvirginica_MBD.sh


#!/bin/bash
/home/sam/software/FastQC/fastqc \
--threads 18 \
--outdir /home/sam/20180409_fastqc_Cvirginica_MBD \
/mnt/owl/nightingales/C_virginica/zr2096_10_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_10_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_1_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_1_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_2_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_2_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_3_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_3_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_4_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_4_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_5_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_5_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_6_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_6_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_7_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_7_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_8_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_8_s1_R2.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_9_s1_R1.fastq.gz \
/mnt/owl/nightingales/C_virginica/zr2096_9_s1_R2.fastq.gz

MultiQC was then run on the FastQC output files.

All files were moved to Owl after the jobs completed.

Results:

FastQC Output folder: 20180409_fastqc_Cvirginica_MBD/

MultiQC Output folder: 20180409_fastqc_Cvirginica_MBD/multiqc_data/

MultiQC report (HTML): 20180409_fastqc_Cvirginica_MBD/multiqc_data/multiqc_report.html

Everything looks good to me.

Steven’s interested in seeing what the trimmed output would look like (and, how it would impact mapping efficiencies). Will initiate trimming.

See the GitHub issue linked above for the full discussion.

DNA Isolation & Quantification – Geoduck larvae metagenome filter rinses

This is another attempt to isolate DNA from two more of the geoduck hatchery metagenome samples Emma delivered on 20180313.

The previous attempt, using DNAzol, did not yield any DNA.

I isolated DNA from the following two samples:

  • MG 5/19 #4
  • MG 5/26 #4

I used the DNA Stool Kit (Qiagen), following the “Stool Human DNA” protocol with the following changes:

  • Incubated @ 95oC for 5mins after initial addition of Buffer ASL. This is a lysis step that might help increase yields (see the “Stool Pathogen Detection” protocol)
  • Did not add InhibitEX Tablet. Deemed unnecessary, since these weren’t stool samples.
  • Eluted in 50μL of Buffer AE

I opted to follow the “Stool Human DNA” protocol, as it processes a larger portion of the initial sample, compared to the “Stool Pathogen Detection” protocol (600μL vs. 200μl)

Samples were quantified using the Roberts Lab Qubit 3.0 with the Qubit High Sensitivity dsDNA Kit (Invitrogen).

10μL of each sample were used.

Results:

Neither sample yielded any detectable DNA. Will discuss with Steven.

Titrations – Yaamini’s Seawater Samples

All data is deposited in the following GitHub repo:

Sample sizes: ~50g

LabX Method:

Daily pH calibration data file:

Daily pH log file:

Titrant batch:

CRM Batch:

Daily CRM data file:

Sample data file(s):

See metadata file for sample info (including links to master samples sheets):

Titrations – Yaamini’s Seawater Samples

All data is deposited in the following GitHub repo:

Sample sizes: ~50g

LabX Methods:

Daily pH calibration data file:

Daily pH log file:

Titrant batch:

CRM Batch:

Daily CRM data file:

Sample data file(s):

See metadata file for sample info (including links to master samples sheets):

Data Received – Crassostrea virginica MBD BS-seq from ZymoResearch

Received the sequencing data from ZymoResearch for the <em>Crassostrea virginica</em> gonad MBD DNA that was sent to them on 20180207 for bisulfite conversion, library construction, and sequencing.

Gzipped FASTQ files were:

  1. downloaded to Owl/nightingales/C_virginica
  2. MD5 checksums verified
  3. MD5 checksums appended to the checksums.md5 file
  4. readme.md file updated
  5. Updated nightingales Google Sheet

Here’s the list of files received:

zr2096_10_s1_R1.fastq.gz
zr2096_10_s1_R2.fastq.gz
zr2096_1_s1_R1.fastq.gz
zr2096_1_s1_R2.fastq.gz
zr2096_2_s1_R1.fastq.gz
zr2096_2_s1_R2.fastq.gz
zr2096_3_s1_R1.fastq.gz
zr2096_3_s1_R2.fastq.gz
zr2096_4_s1_R1.fastq.gz
zr2096_4_s1_R2.fastq.gz
zr2096_5_s1_R1.fastq.gz
zr2096_5_s1_R2.fastq.gz
zr2096_6_s1_R1.fastq.gz
zr2096_6_s1_R2.fastq.gz
zr2096_7_s1_R1.fastq.gz
zr2096_7_s1_R2.fastq.gz
zr2096_8_s1_R1.fastq.gz
zr2096_8_s1_R2.fastq.gz
zr2096_9_s1_R1.fastq.gz
zr2096_9_s1_R2.fastq.gz

Here’s the sample processing history:

Titrations – Hollie’s Seawater Samples

All data is deposited in the following GitHub repo:

Sample sizes: ~50g

LabX Method:

Daily pH calibration data file:

Daily pH log file:

Titrant batch:

CRM Batch:

Daily CRM data file:

Sample data file(s):

See metadata file for sample info (including links to master samples sheets):

Titrations – Hollie’s Seawater Samples

All data is deposited in the following GitHub repo:

Sample sizes: ~50g

LabX Method:

Daily pH calibration data file:

Daily pH log file:

Titrant batch:

CRM Batch:

Daily CRM data file:

Sample data file(s):

See metadata file for sample info (including links to master samples sheets):

DNA Isolation & Quantification – Geoduck larvae metagenome filter rinses

Isolated DNA from two of the geoduck hatchery metagenome samples Emma delivered on 20180313 to get an idea of what type of yields we might get from these.

  • MG 5/15 #8
  • MG 5/19 #6

As mentioned in my notebook entry upon receipt of these samples, I’m a bit skeptical will get any sort of recovery, based on sample preservation.

Isolated DNA using DNAzol (MRC, Inc.) in the following manner:

  1. Added 1mL of DNAzol to each sample; mixed by pipetting.
  2. Added 0.5mL of 100% ethanol; mixed by inversion.
  3. Pelleted DNA 5,000g x 5mins @ RT.
  4. Discarded supernatants.
  5. Wash pellets (not visible) with 1mL 75% ethanol by dribbling down side of tubes.
  6. Pelleted DNA 5,000g x 5mins @ RT.
  7. Discarded supernatants and dried pellets for 5mins.
  8. Resuspended DNA in 20uL of Buffer EB (Qiagen).

Samples were quantified using the Roberts Lab Qubit 3.0 with the Qubit High Sensitivity dsDNA Kit (Invitrogen).

5uL of each sample were used.

Results:

As expected, both samples did not yield any detectable DNA.

Will discuss with Steven on what should be done with the remaining samples.