Sample sheet (Google Sheet):
Samples were stored in -80oC:
Sample sheet (Google Sheet):
Samples were stored in -80oC:
Received and stored @-80C in rack 8, row 5, column 5.
The following information was sent with the samples:
Sample.ID | Date | Temp | pCO2 | Notes |
---|---|---|---|---|
031 | 26-Aug-2016 | 15 | 400 | |
032 | 26-Aug-2016 | 15 | 400 | |
033 | 26-Aug-2016 | 15 | 400 | |
034 | 26-Aug-2016 | 15 | 400 | |
035 | 26-Aug-2016 | 15 | 400 | All sample sent; it will be in 2mL screw-cap vial |
036 | 26-Aug-2016 | 15 | 400 | |
103 | 26-Aug-2016 | 15 | 2800 | |
104 | 26-Aug-2016 | 15 | 2800 | |
105 | 26-Aug-2016 | 15 | 2800 | |
106 | 26-Aug-2016 | 15 | 2800 | All sample sent; it will be in 2mL screw-cap vial |
108 | 26-Aug-2016 | 15 | 2800 |
Katie sent this additional info in an email to Steven and me:
These C. virginica samples were exposed to control (400, 6 samples) and OA (2800, 5 samples) conditions for ~4 weeks at 15C. Gonad was carefully extracted by peeling back the outer membrane, flash frozen in liquid N, and placed in -80C (until today when we removed it). During sampling, it was difficult to get a lot of what we considered “pure” gonadal tissue. We sent you ~1/2 of the amount of tissue we have for all samples except for the two samples which were very low and we sent you all the tissue sample we have. Each should be about 10-20 mg of tissue, which I’m worried is not enough for MBD-BS seq. Fingers crossed.
Received notice the sequencing data was ready from Genewiz for the samples submitted 20151222.
Download the FASTQ files from Genewiz project directory:
wget -r -np -nc -A "*.gz" ftp://username:password@ftp2.genewiz.com/Project_BS1512183
Since two species were sequenced (C.gigas & O.lurida), the corresponding files are in the following locations:
http://owl.fish.washington.edu/nightingales/O_lurida/
http://owl.fish.washington.edu/nightingales/C_gigas/
In order to process the files, I needed to identify just the FASTQ files from this project and save the list of files to a bash variable called ‘bsseq':
bsseq=$(ls | grep '^[0-9]\{1\}_*' | grep -v "2bRAD")
Explanation:
bsseq=
$(ls | grep '^[0-9]\{1\}_*' | grep -v "2bRAD")
FILENAME | SAMPLE NAME | SPECIES |
1_ATCACG_L001_R1_001.fastq.gz | 1NF11 | O.lurida |
2_CGATGT_L001_R1_001.fastq.gz | 1NF15 | O.lurida |
3_TTAGGC_L001_R1_001.fastq.gz | 1NF16 | O.lurida |
4_TGACCA_L001_R1_001.fastq.gz | 1NF17 | O.lurida |
5_ACAGTG_L001_R1_001.fastq.gz | 2NF5 | O.lurida |
6_GCCAAT_L001_R1_001.fastq.gz | 2NF6 | O.lurida |
7_CAGATC_L001_R1_001.fastq.gz | 2NF7 | O.lurida |
8_ACTTGA_L001_R1_001.fastq.gz | 2NF8 | O.lurida |
9_GATCAG_L001_R1_001.fastq.gz | M2 | C.gigas |
10_TAGCTT_L001_R1_001.fastq.gz | M3 | C.gigas |
11_GGCTAC_L001_R1_001.fastq.gz | NF2_6 | O.lurida |
12_CTTGTA_L001_R1_001.fastq.gz | NF_18 | O.lurida |
I wanted to add some information about the project to the readme file, like total number of sequencing reads generated and the number of reads in each FASTQ file.
Here’s how to count the total of all reads generated in this project
totalreads=0; for i in $bsseq; do linecount=`gunzip -c "$i" | wc -l`; readcount=$((linecount/4)); totalreads=$((readcount+totalreads)); done; echo $totalreads
Total reads = 138,530,448
C.gigas reads: 22,249,631
O.lurida reads: 116,280,817
Code explanation:
totalreads=0;
for i in $bsseq;
do linecount=
`gunzip -c "$i" | wc -l`;
readcount=$((linecount/4));
totalreads=$((readcount+totalreads));
done;
echo $totalreads
Next, I wanted to generate list of the FASTQ files and corresponding read counts, and append this information to the readme file.
for i in $bsseq; do linecount=`gunzip -c "$i" | wc -l`; readcount=$(($linecount/4)); printf "%s\t%s\n%s\t\t\n" "$i" "$readcount" >> readme.md; done
Code explanation:
for i in $bsseq; do linecount=`gunzip -c "$i" | wc -l`; readcount=$(($linecount/4));
printf "%s\t%s\n\n" "$i" "$readcount" >> readme.md;
>> readme.md; done
Pooled 10ng of each of the libraries prepared yesterday with TruSeq DNA Methylation Library Kit (Illumina) for sequencing at Genewiz.
SAMPLE | VOLUME FOR 10ng (μL) |
1NF11 | 4.13 |
1NF15 | 5.32 |
1NF16 | 3.65 |
1NF17 | 3.94 |
2NF4 | 3.68 |
2NF5 | 4.10 |
2NF6 | 4.20 |
2NF7 | 5.32 |
M2 | 4.59 |
M3 | 3.91 |
NF2_6 | 4.00 |
NF_18 | 3.76 |
Samples were sent to Genewiz on dry ice via standard overnight FedEx.
Re-quantified the libraries that were completed yesterday using the Qubit3.0 dsDNA HS (high sensitivity) assay because the library concentrations were too low for the normal broad range kit.
Results:
Qubit Quants and Library Normalization Calcs: 20151222_qubit_illumina_methylation_libraries
SAMPLE | CONCENTRATION (ng/μL) |
1NF11 | 2.42 |
1NF15 | 1.88 |
1NF16 | 2.74 |
1NF17 | 2.54 |
2NF5 | 2.72 |
2NF6 | 2.44 |
2NF7 | 2.38 |
2NF8 | 1.88 |
M2 | 2.18 |
M3 | 2.56 |
NF2_6 | 2.5 |
NF_18 | 2.66 |
Things look pretty good. The TruSeq DNA Methylation Library Kit (Illumina) suggests that the libraries produced should end up with concentrations >3ng/μL, but we have plenty of DNA here to make a pool for running on the HiSeq2500.
Following the guidelines of the TruSeq DNA Methylation Library Prep Guide (Illumina), I ran 1μL of each sample on an RNA Pico 6000 chip on the Seeb Lab’s Bioanalyzer 2100 (Agilent) to confirm that bisulfite conversion from earlier today worked.
Results:
Data File 1(Bioanlyzer 2100): 2100 expert_Eukaryote Total RNA Pico_DE72902486_2015-12-18_21-05-04.xad
Data File 1(Bioanlyzer 2100): 2100 expert_Eukaryote Total RNA Pico_DE72902486_2015-12-18_21-42-55.xad
Firstly, the ladder failed to produce any peaks. Not sure why this happened. Possibly not denatured? Seems unlikely, but next time I run the Pico assay, I’ll denature the ladder aliquot I use prior to running.
Overall, the samples look as they should (see image from TruSeq DNA Methylation Kit manual below), albeit some are a bit lumpy.
After confirming that the DNA available for this project looked good, I performed bisulfite treatment on the following gDNA samples:
Sample names breakdown like this:
1NF#
1 = Fidalgo Bay outplants
NF = Fidalgo Bay broodstock origination
# = Sample number
2NF#
Same as above, but:
2 = Oyster Bay outplants
NF2_# (Oysters grown in Oyster Bay; DNA provided by Katherine Silliman)
NF2 = Fidalgo Bay broodstock origination, family #2
# = Sample number
M2/M3 = C.gigas from Katie Lotterhos
Followed the guidelines of the TruSeq DNA Methylation Library Prep Guide (Illumina).
Used the EZ DNA Methylation-Gold Kit (ZymoResearch) according to the manufacturer’s protocol with the following changes/notes:
DNA quantity calculations are here (Google Sheet): 20151218_oly_bisulfite_calcs
Samples were stored @ -20C. Will check samples via Bioanalyzer before proceeding to library construction.