Sam’s Notebook:TrimGalore/FastQC/MultiQC – Trim 10bp 5’/3′ ends C.virginica MBD BS-seq FASTQ data

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Steven found out that the Bismarck documentation (Bismarck is the bisulfite aligner we use in our BS-seq pipeline) suggests trimming 10bp from both the 5′ and 3′ ends. Since this is the next step in our pipeline, we figured we should probably just follow their recommendations!

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:

Hey! Look at that! Everything is much better! Thanks for the excellent documentation and suggestions, Bismarck!

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Sam’s Notebook:DNA Isolation & Quantification – Metagenomics Water Filters

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Isolated DNA from the following two filters:

20180411_metagenome_filters.jpg

DNA was isolated with the DNeasy Blood & Tissue Kit (Qiagen), following a modified version of the Gram-Positive Bacteria protocol:

  • filters were unfolded and unceremoniously stuffed into 1.7mL snap cap tubes
  • did not perform enzymatic lysis step
  • filters were incubated with 400μL of Buffer AL and 50μL of Proteinase K (both are double the volumes listed in the kit and are necessary to fully coat the filter in a 1.7mL snap cap tube)
  • 56oC incubations were performed overnight
  • 400μL of 100% ethanol was added to each after the 56oC incubation
  • samples were eluted in 50μL of Buffer AE
  • all spins were performed at 20,000g

Samples were quantified with the Roberts Lab Qubit 3.0 and the Qubit 1x dsDNA HS Assay Kit.

Used 10μL of each sample for measurement (see Results for update).

Results:

Raw data (Google Sheet): 20180411_qubit_metagenomics_filters

Sample Concentration(ng/μL) Initial_volume(μL) Yield(ng)
filter 5/22 #7 pH8.2 20.8 50 1040
filter 5/26 #7 pH8.2 11.6 50 580

NOTE: For “filter 5/22 #7 pH8.2″ the initial quantification using 10μL ended up being too concentrated. Re-ran using 5μL.

Both samples have yielded DNA. This is, obviously, an improvement over the previous attempts to isolate DNA from ammonium bicarbonate filter rinses that Emma supplied me with.

Will discuss with Steven and get an idea of which filters to isolate additional DNA from.

Samples were stored Sam gDNA Box #2, positions G6 & G7. (FTR 213, #27 (small -20oC frezer)

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Sam’s Notebook:TrimGalore/FastQC/MultiQC – 2bp 3′ end Read 1s Trim C.virginica MBD BS-seq FASTQ data

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Earlier today, I ran TrimGalore/FastQC/MultiQC on the Crassostrea virginica MBD BS-seq data from ZymoResearch and hard trimmed the first 14bp from each read. Things looked better at the 5′ end, but the 3′ end of each of the READ1 seqs showed a wonky 2bp blip, so decided to trim that off.

I ran TrimGalore (using the built-in FastQC option), with a hard trim of the last 2bp of each first read set that had previously had the 14bp hard trim 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:

Well, this is a bit strange, but the 2bp trimming on the read 1s looks fine, but now the read 2s are weird in the same region!

Regardless, while this was running, Steven found out that the Bismarck documentation (Bismarck is the bisulfite aligner we use in our BS-seq pipeline) suggests trimming 10bp from both the 5′ and 3′ ends. So, maybe this was all moot. I’ll go ahead and re-run this following the Bismark recommendations.

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Grace’s Notebook: April 10, 2018 Crab Project Updates

Qubit and RNA isolation

I ran Qubit yesterday on 14 tubes that I had previously isolated RNA from. The results are: Here and Here

Today, I ran Qubit on two samples that I isolated RNA from in the early stages of this process. This means that the protocol I used was different than the one I currently am using. There are less centrifuge and mixing steps. However, I ran them on the Qubit because I somehow ended up choosing those two crabs as part of the subset of surviving crabs for my current RNA isolation samples.

To demonstrate a little more clearly what I mean: Below is the subset of hemolymph samples I chose to isolate RNA from:
img

The two samples (88 and 36) highlighted are samples that I tried out the protocol on a little over a month ago or so.

I had not intended to select samples that I had already isolated for the subset, but I mistakenly did. The Qubit results were good, though.
Tube 36 –> 4.16 ng/µL
Tube 88 –> 2.81 ng/µL

I am going to have to ask Sam and/or Steven about whether I should pick a different set of samples becuase the RNA isolation protocol was slightly different between what I used on tube 88 and 36 and what I am currently using.

DecaPod

I published episode 1 FINALLY! Pam and I sat down at the end of last week’s crab meeting #2 to record a little background info.

Now that that is finally finished, I am going to start editing the other recordings I have and release one next week, and the other the following week.

I have the recording of Crab Mtg #2 (next week’s podcast)
and another of Pam answering some of my questions about the experimental design as well as some questions that others have asked me and I was unable to answer.

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