Sequencing Data – LSU C.virginica MBD BS-Seq

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:

Sample Treatment Barcode
HB2 25,000ppm oil ATCACG
HB16 25,000ppm oil TTAGGC
HB30 25,000ppm oil TGACCA
NB3 No oil ACAGTG
NB6 No oil GCCAAT
NB11 No oil CAGATC

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

Bisulfite NGS Library Prep – Bisulfite Conversion & Illumina Library Construction of C.gigas larvae DNA

Bisulfite Conversion

The previous attempt at constructing a library for the 1000ppm larvae samples failed. I had previously sheared, quantified, and concentrated the DNA from this sample. As I had done previously, I combined 50ng from each of the two 1000ppm samples for a total of 100ng, and brought the sample volume up to 24μL with NanoPure H2O.

Bisulfite conversion was performed with the Methylamp DNA Modification Kit (Epigentek) according to the manufacturer’s protocol.

Sample was eluted with 10μL of Buffer R6 for immediate use.

 

Library Prep

Bisulfite Illumina library was made with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • Skipped Step 7.1 (per manufacturer’s recommendation for samples starting with <200ng)
  • Ran 13 cycles during the library amplification step (per manufacturer’s recommendation for samples starting with 100ng)

Sample was transferred to 1.5mL snap cap tube and stored @ -20C.  Will quantify library on Monday when Jake is also finished with his 12 libraries.

 

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite library quantification

The two completed BS Illumina libraries (400ppm and 1000ppm) were quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of  each sample and the standards.  All standards were run in triplicate.  Due to limited sample, the two libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek).

 

Results:

The standard curve, raw fluorescence, and calculated concentrations (as determined by the Gen5 (BioTek) software) can be seen here: 20150128_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

The standard curve was excellent, exhibiting a R² value = 0.999

 

Sample Concentration (ng/uL)
400ppm 10.592
1000ppm 0.0

 

The 400ppm library looks great, with a good yield.

The 1000ppm library appears to have no measurable quantity of DNA in it.  This is surprising, and disconcerting, as both samples were processed in parallel.  As such, there should be virtually no difference between them, in regards to the library construction process and subsequent yields.

To verify that this wasn’t a pipetting error on my part, I re-quantified the 1000ppm library (in duplicate) and still no detectable DNA.

Will repeat the bisulfite conversion and library construction process on the 1000ppm sample in order to generate a usable library for sequencing.

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite DNA (continued from yesterday)

Continued Illumina library prep of bisulfite-treated DNA samples (400ppm and 1000ppm; from 20150114)  with Methylamp DNA Modification Kit (Epigentek). Performed bead clean up immediately after End Repair.

PCR cycles: 14

No other changes were made to the manufacturer’s protocol.

Epigentek Barcode Indices assigned, per their recommendations for using two libraries for multiplexing:

400ppm – barcode #6 – GCCAAT

1000ppm – barcode #12 – CTTGTA

The two libraries were stored @ -20C and will be quantified tomorrow.

 

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite DNA

The two pooled bisulfite-treated DNA samples (400ppm and 1000ppm) from 20150114 were used to prepare bisulfite Illumina libraries with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • Stopped after End Repair step (prior to magnetic bead clean up).  Samples stored @ -20C

Library Cleanup – LSU C.virginica MBD BS Library

I was contacted by the sequencing facility at the University of Oregon regarding a sample quality issue with our library.  As evidenced by the electropherogram below, there is a great deal of adaptor primer dimer (the peak at 128bp):

 

This is a problem because such a high quantity of adaptor sequence will result in the majority of reads coming off the Illumina being just adaptor sequences.

With the remainder of the library sample prepared earlier, I performed the recommended clean up procedure for removing adaptor sequences in the EpiNext Post-Bisulfite DNA Library Preparation Kit – Illumina (Epigentek).    Briefly:

  • Brought sample volume up to 20uL with NanoPure H2O (added 9.99uL)

  • Added equal volume of MQ Beads

  • Washed beads 3x w/80% EtOH

  • Eluted DNA w/12uL Buffer EB (Qiagen)

After clean up, quantified the sample via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of the sample and the standards.  All standards were run in duplicate and read on a FLx800 plate reader (BioTek).

Results are here: 20150122 – LSU_virginicaMBDlibraryCleanup

Library concentration = 2.46ng/uL

Brought the entire sample up to 20uL with Buffer EB (Qiagen) and a final concentration of 0.1% Tween-20 (required by the sequencing facility).

Sent sample to the University of Oregon to replace our previous submission.

DNA Bisulfite Conversion – C.gigas larvae OA Sheared DNA

After discussing with Steven, decided to use only samples from 20110513, due to high algae amounts present in the 20110519 samples.

Pooled the DNA of the 400ppm samples (6B2 & 6B5) and pooled the DNA of the 1000ppm samples(1B1 & 1B2) , for a total of two samples. 50ng of each sample was used to make the pools, for a toal of 100ng of DNA in each pool. Calculations are below:
https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

Each pooled sample was brought up to a final volume of 24μL for use in bisulfite conversion kit.

Performed bisulfite conversion of sheared DNA from 20150113 using the Methylamp DNA Modification Kit (Epigentek) according the manufacturer’s protocol.

Samples were eluted with 10μL of Buffer R6 and stored @ -20C.

 

SpeedVac – C.gigas larvae OA DNA

The DNA extracted and sheared on 20150109 was in a volume too great to proceed with bisulfite conversion.  Dried the samples to complete dryness in a SpeedVac (~4hrs).  Reconsitituted DNA in 24μL of NanoPure water.  Will bisulfite convert tomorrow.

Sample list:

SAMPLE ID DATE TREATMENT (ppm) # LARVAE TOTAL DNA (ng)
6B5 20110513 400 5,000 105
1B2 20110513 1000 5,000 109
6B2 20110513 400 10,000 117
1B1 20110513 1000 10,000 593
1B1 20110519 1000 NA 500
1B2 20110519 1000 NA 500
6B2 20110519 400 NA 269
6B1 20110519 400 NA 345

 

Updated the quantification spreadsheet with a column labeled “[New](ug/uL)” that calculates the new concentrations of the above samples in the 24μL volume.

https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

 

DNA Isolation – C.gigas larvae from 2011 NOAA OA Experiment

DNA was isolated from the following samples:

SAMPLE ID DATE TREATMENT (ppm) # LARVAE
6B5 20110513 400 5,000
1B2 20110513 1000 5,000
6B2 20110513 400 10,000
1B1 20110513 1000 10,000
1B1 20110519 1000 NA
1B2 20110519 1000 NA
6B2 20110519 400 NA
6B1 20110519 400 NA

 

Some tubes contained a high quantity of algae, based on quantity of material in tube and overall green color.

Samples 1B1 & 1B2 from 20110519 have excessive quantities of algae.

Samples 6B1 & 6B1 from 20110519 have a fair amount of algae.

See pic:

 

Sample tubes after brief spin, prior to DNA isolation.

Prior to isolation, samples were briefly spun (12,000g, 15s @ RT). Supernatants were discarded.

 

 

DNA Isolation

DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen).

Samples were resuspended in 180uL of Buffer AL and 20uL of Proteinase K. Samples were mixed by vortexing and incubated @ 56C O/N.

The manufacturer’s protocol (Purification of Total DNA from Animal Tissues (Spin-Column Protocol)) was followed.

Due to low quantities of starting tissue, samples were eluted with 200μL of Buffer EB to maximize DNA recovery.

 

DNA Quantification

Samples were prepared for quantification via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen). The manufacturer’s protocol was altered to use 5μL of sample and 5μL of standards (instead of 10μL) in each well. All samples/standards were run in duplicate and read on a FLx800 plate reader (BioTek).

Mean fluorescence of the standards were plotted with a best-fit line. The resulting equation from the best-fit line was used to determine sample concentrations from their mean fluorescence.

 

Results:

Calcs and resulting quantities are here:

https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

 

All samples have yields great enough to proceed with shearing and bisulfite conversion.

Samples 1B1 and 1B2 from 20110519 have extremely large yields.  This is not surprising, considering the amount of algae present in the source tubes.  Will process only 500ng from each sample.

 

 

DNA Shearing

Adjusted volume of all samples to 190μL using Buffer EB (Qiagen) in 1.5mL snap-cap tubes.

Samples were sonicated/sheared in the Bioruptor (Diagenode) with the following cycling protocol:

25 cycles of:

30s on

30s off

Cycling params were adjusted from the last time I performed this, since I felt the final sheared size was a bit on the small size.

After shearing, samples were stored @ 4C until I could SpeedVac them to reduce their volumes, as the bisulfite treatment step requires volumes < 24uL.

 

Bisulfite NGS Library – LSU C.virginica Oil Spill MBD Bisulfite DNA Sequencing Submission

Combined the following libraries in equal quantities (17ng each) to create a single, multiplexed sample for sequencing (LSU_Oil_01):

  • HB2 – 1 (ATCACG)
  • HB16 – 3 (TTAGGC)
  • HB30 – 4 (TGACCA)
  • NB3 – 5 (ACAGTG)
  • NB6 – 6 (GCCAAT)
  • NB11 – 7 (CAGATC)

Quantified pooled libraries using the Quant-iT dsDNA BR Kit (Invitrogen) with a FLx800 plate reader (BioTek). Used 1μL of the pooled sample, run in duplicate. Used 1uL of standards, run in duplicate.

Results:

pooled libraries = 6.575ng/μL

Will submit to University of Oregon Genomics Core Facility for 100bp, single end Illumina HiSeq2500 sequencing. They need 10nM of sample. For a library with average size range of 300-400bp, this requires a sample volume of 20uL with a concentration of 2.28ng/μL in a solution of 0.1% Tween20 in Buffer EB (Qiagen).

Combined 6.94μL of pooled libraries with 13.06 of 0.1% Tween20/EB solution.

Submitted sample LSU_Oil_01 to University of Oregon Genomics Core Facility via O/N FedEx on dry ice. Sample was assigned order # 2112.