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:

• Tissue received from K. Lotterhos
• DNA isolation
• Yaamini MBD Day 1
• Yaamini MBD Day 2
• Yaamini MBD Day 3
• Yaamini MBD Day 4
• EtOH precip, quantification, shipped

Sent Crassostrea virginica samples to Qiagen, as part of the collaboration we have with them for testing their new bisulfite conversion kit on various reduced representation DNA.

Samples were sent on dry ice via FedEx International Priority: 771231112481

Here are the samples I sent:

gDNA C.virginica – Genomic DNA, 20uL, 58.4ng/uL
MBD 1 virginica – Fragmented (~400bp average size), MBD-enriched, 25uL, 18.3ng/uL
MBD 2 virginica – Fragmented (~400bp average size), MBD-enriched, 25uL, 19.6ng/uL
MspI 1 virginica – gDNA digested with MspI, 25uL, 53.4ng/uL
MspI 2 virginica – gDNA digested with MspI, 25uL, 31.0ng/uL

Genomic DNA was isolated from mantle tissue using the E.Z.N.A. Mollusc DNA Kit (Omega). DNA was eluted with the Elution Buffer supplied with the kit.

MBD enrichment was performed using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). DNA was resuspended in Buffer EB (Qiagen).

MspI digestions were performed using MspI (NEB) and were subjected to a phenol:chloroform cleanup, post-digestion. DNA was resuspended in Buffer EB (Qiagen).

No tests have been performed on the samples to evaluate the presence/absence of non-oyster DNA.

Reference genome is available here: https://www.ncbi.nlm.nih.gov/assembly/GCF_002022765.2/

Notebook entries:

gDNA Isolation:

http://onsnetwork.org/kubu4/2017/12/11/dna-isolation-quantification-crassotrea-virginica-mantle-gdna/

MBD:

DNA Shearing and Bioanalyzer assessments: http://onsnetwork.org/kubu4/2017/12/11/dna-sonication-bioanalzyer-c-virginica-gdna-for-medip/
Day 1: http://onsnetwork.org/kubu4/2018/01/08/mbd-enrichment-crassostrea-virginica-sheared-dna-day-1/
Day 2: http://onsnetwork.org/kubu4/2018/01/09/mbd-enrichment-crassostrea-virginica-sheared-dna-day-2/
Day 3: http://onsnetwork.org/kubu4/2018/01/10/mbd-enrichment-crassostrea-virginica-sheared-dna-day-3/
Quants: http://onsnetwork.org/kubu4/2018/01/10/dna-quantification-c-virginica-mbd-enriched-dna/

MspI Digestion:

Digestion: http://onsnetwork.org/kubu4/2018/01/11/restriction-digestion-mspi-on-crassotrea-virginica-gdna/
Phenol:Chloroform cleanup: http://onsnetwork.org/kubu4/2018/01/11/phenolchloroform-extractions-and-etoh-precipitations-mspi-digestions-of-c-virginica-dna-from-earlier-today/
Quants: http://onsnetwork.org/kubu4/2018/01/11/dna-quantification-mspi-digested-crassostrea-virginica-gdna/

Continued MBD enrichment of C.virginica DNA from yesterday for Qiagen project.

Followed the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) manufacturer’s protocol for input DNA amounts of 1 -10ug (I am using 8ug in each of two samples).

Since the protocol has two elution steps that are each saved separately from each other for each sample, I did the following to combine the two elution fractions into a single sample:

• Pelleted one elution fraction from each sample
• Discarded supernatant from pelleted sample
• Transferred second elution fraction to the pellet from the first elution fraction
• Pelleted second elution fraction

The rest of the ethanol precipitation procedure was followed per the manufacturer’s protocol.

Final pellets were resuspended in 25μL of Buffer EB (Qiagen) and stored temporarily on ice for quantification.

Continued MBD enrichment for C.virginica and Qiagen project from yesterday.

Followed the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) manufacturer’s protocol for input DNA amounts of 1 -10ug (I am using 8ug in each of two samples).

Performed a single, high-salt elution.

Samples were precipitated O/N @ -80C.

We opted to go with ZymoResearch for this project because they could do the bisulfite treatment and finish the sequencing by the end of January.

Submitted the following 18 Ostrea lurida MBD-enriched gDNA samples to ZymoResearch for bisulfite treatment and subsequent Illumina sequencing (50bp, single read):

The samples will be bisulfite treated, Illumina libraries constructed, multiplexed, and this multiplexed library will be sequenced three times.

Quantified the MBD enriched samples prepped over the last two days: MBD enrichment, EtOH precipiation.

Samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

Google Sheet: 20151123_MBD_libraries_quantification

Standard curve looked good – R² = 0.999

MBD recovery ranged from ~250 – 600ng.

MBD percent recoveries ranged from ~2 – 20%. Input DNA quantities were taken from Katherine’s numbers (Google Sheet): Silliman-DNA-Samples

Will contact services about getting bisulfite Illumina sequencing performed.

Our sequencing data (Illumina HiSeq2500, 100SE) for this project has completed by Univ. of Oregon Genomics Core Facility (order number 2112).

Samples sequenced/pooled for this run:

All code listed below was run on OS X 10.9.5

Downloaded all 15 fastq.gz files to Owl/web/nightingales/C_virginica:

$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_001.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_002.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_003.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_004.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_005.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_006.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_007.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_008.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_009.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_010.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_011.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_012.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_013.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_014.fastq.gz
$curl -O http://gcf.uoregon.edu:8080/job/download/2112?fileName=lane1_NoIndex_L001_R1_015.fastq.gz

Renamed all files by removing the beginning of each file name (2112?fileName=) and replacing that with 2112_:

$for file in 2112*lane1_NoIndex_L001_R1_0*; do mv "$file" "${file/#2112?fileName=/2112_}"; done

Created a directory readme.md (markdown) file to list & describe directory contents: readme.md

$ls *.gz >> readme.md

Note: In order for the readme file to appear in the web directory listing, the file cannot be all upper-case.

Created MD5 checksums for each fastq.gz file: checksums.md5

$md5 *.gz >> checksums.md5