Geoduck RRBS Library Prep

Prepping RRBS libraries for geoduck juvenile OA acclimatization study.

20161201

Chose 12 samples from a single time point (Day 10, 3 treatments * 4 replicates) for the first sequencing library preps. 100ng was used as the starting amount of DNA for each sample and the volume of water in each reaction was adjusted as necessary. 0.5% unmethylated lambda DNA was spiked in (w/w DNA) to determine conversion efficiency by estimating the error rate at which a C count occurs at an unmethylated C position

Step 1 MSPI DNA Cutting

MSPI restriction endonuclease in NEBuffer2

  • MspI - 25,000U (NEB cat: R0106L)
  • NEBuffer2 10x (NEB cat: B7002S)

Reaction Mix

  • Samples were mixed as follows to a total reaction volume of 30µl. Samples were incubated at 37°C starting at 15:00
  • Lambda DNA (581ng/µl) was diluted 1:1160 for a concentration of 0.5ng/µl and 1µl added to each sample
  • Unmethylated lamda phage DNA (Promega cat: D1501)

    • 3µl NEBuffer2
    • 1µl MSPI 20U/µl
    • 16µl DNA (100ng)
    • 10µl of H20
    • 30µl total
EPI.Tube.Num Tank Treatment DNA.Extraction DNA.Conc.ng.µl vol.for.100ng lambda.0.5% Water.µl NSBuffer2.µl MSPI.µl
EPI_103 Tank11 Low 20161028 18.3 5.5 1 19.5 3 1
EPI_104 Tank11 Low 20161007 9.28 10.8 1 14.2 3 1
EPI_111 Tank12 Super.Low 20161007 55.6 1.8 1 23.2 3 1
EPI_113 Tank12 Super.Low 20161007 18.8 5.3 1 19.7 3 1
EPI_119 Tank13 Ambient 20161007 17.6 5.7 1 19.3 3 1
EPI_120 Tank13 Ambient 20161007 16.5 6.1 1 18.9 3 1
EPI_127 Tank14 Low 20161004 35.2 2.8 1 22.2 3 1
EPI_128 Tank14 Low 20161004 29.8 3.4 1 21.6 3 1
EPI_135 Tank15 Ambient 20161004 20.2 5 1 20 3 1
EPI_136 Tank15 Ambient 20161004 18.2 5.5 1 19.5 3 1
EPI_143 Tank16 Super.Low 20161028 41.2 2.4 1 22.6 3 1
EPI_145 Tank16 Super.Low 20161007 9.64 10.4 1 14.6 3 1

Step 1 conclusions

  • no errors were seen on the PCR machine. Samples assumed to have been cut with MSPI

Step 2 Bisulfite conversion

Samples

Added additional samples to compare RRBS above with whole genome library preps

EPI.Tube.Num Tank Treatment DNA.Extraction DNA.Conc.ng.µl vol.for.100ng lambda.0.5% Water.µl
EPI_111 Tank12 Super.Low 20161007 55.6 1.8 1 27.2
EPI_128 Tank14 Low 20161004 29.8 3.4 1 25.6
EPI_135 Tank15 Ambient 20161004 20.2 5 1 24.0
  • EPI_111 - Whole Genome sample, no MSPI cutting
  • EPI_128 - Whole Genome sample, no MSPI cutting
  • EPI_135 - Whole Genome sample, no MSPI cutting

Bisulfite conversion kit prep

  • Prepared the CT Conversion Reagent for larger DNA samples (30µl) by adding 800µl of water, 300µl of M-Dilution and 50µl of M-Dissolving

  • Added 30µl of sample described in table to 120 µl of CT Conversion Reagent, mixed by flicking, and spun down
  • Placed tubes in PCR machine at 10:45 and set as follows:
    • 98°C for 10 min
    • 64°C for 2.5 hours
    • 4°C forever
  • followed protocol steps EZ DNA Methylation-Gold Manual

  • Eluted in 12µl of elution buffer

  • 1µl was used to quantify the samples on the nanodrop
Sample.ID ng/µl
EPI_103 18.06
EPI_104 19.39
EPI_111 19.43
EPI_113 18.76
EPI_119 18.83
EPI_120 20.48
EPI_127 21.31
EPI_128 22.01
EPI_135 21.56
EPI_136 20.35
EPI_143 19.92
EPI_145 19.99
EPI_111 WG 23.04
EPI_128 WG 23.69
EPI_135 WG 23.63
  • 1µl was used to assess fragment size on the bioanalyzer.

Bisulfite converted ssDNA assessment

Expected DNA size for Gold kit (A)

Expected DNA size

Results of bisulfite converted samples

Chip 1 Data

Chip1

Chip 2 Data

Chip2

  • size quantification on sample 111-wg does not seem to be displaying correctly
  • Gel marker is offset
  • gel offset

Step 2 conclusions

  • All samples are of approptiate size to continue with library prep

Step 3 Illumina Library Prep

Ilumina TruSeq DNA Methylation Library Preparation Guide

Illumina Adapter Sequences Document # 1000000002694 v01 17 February 2016 TruSeq DNA Methylation Index PCR Primers 5’ CAAGCAGAAGACGGCATACGAGAT[6 bases]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

  • Index 1: ATCACG
  • Index 2: CGATGT
  • Index 3: TTAGGC
  • Index 4: TGACCA
  • Index 5: ACAGTG
  • Index 6: GCCAAT
  • Index 7: CAGATC
  • Index 8: ACTTGA
  • Index 9: GATCAG
  • Index 10: TAGCTT
  • Index 11: GGCTAC
  • Index 12: CTTGTA

Illumina library prep was completed according to manufacturer’s instructions with modifications as described in red text on the protocol and listed below.

  • samples eluted in 12µl post BS conversion
  • 200µl of 80% ethanol used for rinsing beads
  • Samples were stored at -20 for ~24h between Purify the Tagged DNA and Amplify the Library and Add an Index steps
  • Library prep was started 20161202
  • Library prep was finished 20121204
  • Individual barcodes were used for each sample from the Illumina TruSeq DNA Methylation Index PCR Primers (10 reactions, 12 indexes Cat #: EGIDX81312)
Sample.ID Index.Number Index.Sequence
EPI_103 1 ATCACG
EPI_104 2 CGATGT
EPI_111 3 TTAGGC
EPI_113 4 TGACCA
EPI_119 5 ACAGTG
EPI_120 6 GCCAAT
EPI_127 7 CAGATC
EPI_128 8 ACTTGA
EPI_135 9 GATCAG
EPI_136 10 TAGCTT
EPI_143 11 GGCTAC
EPI_145 12 CTTGTA
EPI_111 WG 1 ATCACG
EPI_128 WG 2 CGATGT
EPI_135 WG 3 TTAGGC

Library Quantification

20161205 Libraries were quantified on the Qubit using the dsDNA High Sensitivity Kit

  • Samples were loaded 1µl of library + 199µl of Qubit dye/buffer mix (1:200)
  • Standards were loaded 10µl of standard + 190µl of Qubit dye/buffer mix (1:200)

  • Illumin indicates samples should be >3ng/µl
Sample.ID ng/µl
EPI_103 4.34
EPI_104 4.80
EPI_111 5.46
EPI_113 4.88
EPI_119 5.10
EPI_120 4.58
EPI_127 4.58
EPI_128 4.80
EPI_135 4.08
EPI_136 3.70
EPI_143 2.88
EPI_145 2.92
EPI_111 WG 3.40
EPI_128 WG 4.12
EPI_135 WG 2.24

Library Quality

Libraries were not concentrated enough to measure using Agilent DNA 12000 kit 20161204

  • 1µl of sample was used for each and no data were visible with DNA 12000 kit

20161205

  • 1.0µl of each sample was run on the Agilent DNA High Sensitivity Kit
  • A few diluted DNA samples were also run in extra cells to view original DNA with more resolution

Illumina DNA Methylation kit expected library size on high sensitivity chip expected size

RRBS Chip 1 Data

RRBS Library Chip 1

  • EPI_103
  • EPI_104
  • EPI_111
  • EPI_113
  • EPI_119
  • EPI_120
  • EPI_127
  • EPI_128
  • EPI_135
  • EPI_136
  • EPI_143
  • EPI_1451

RRBS Chip 2

RRBS Library Chip 2

  • EPI_145
  • EPI_111 WG
  • EPI_128 WG
  • EPI_135 WG
  • EPI_103 DNA Diluted 1:1
  • EPI_104 DNA Diluted 1:1
  • EPI_111 DNA Diluted 1:10
  • EPI_113 DNA Diluted 1:1
  • EPI_119 DNA Diluted 1:1
  • EPI_128 DNA Diluted 1:3
  • EPI_135 DNA Diluted 1:3

  • Original DNA samples overloaded the chip sensitivity and data are not useful

RRBS Library Prep Conclusions

  • samples have the correct quantity of ~3ng/µl and more than enough ~15µl (45ng) to load for sequencing
  • library size ranges from 150bp to 1000bp and peaks just around 300bp, which matches the expected range in the kit guide
  • There does not appear to be a difference in library size between whole genome and RRBS preps as seen in the traces of the RRBS chip 2 results
  • libraries appear to be of good quality for sequencing

Library Pooling for seqeuncing submission

Libraries were diluted to 10nM and pooled 2µl each for a pooled sample of 10nM in 24µl

Samples were sent to Genewiz on 20161213

Day Lane Sample.ID Library Concentration (ng/uL) Library Concentration (ug/uL) Library Volume (uL) Molecular Weight pmol/660pg 10^6pg/ug DNA Size 1/N (N = bp) pmol DNA Library Concentration (uM) Library Concentration (nM) Dilution Factor for 10nM Vol Library added µl Vol Elution Buffer µl
Day10 Lane1 EPI_103 4.34 0.0043 15 0.002 1000000 0.0033 0.33 0.022 21.92 0.46 1.8 2.2
Day10 Lane1 EPI_104 4.8 0.0048 15 0.002 1000000 0.0033 0.36 0.024 24.24 0.41 1.7 2.4
Day10 Lane1 EPI_111 5.46 0.0055 15 0.002 1000000 0.0033 0.41 0.028 27.58 0.36 1.5 2.5
Day10 Lane1 EPI_113 4.88 0.0049 15 0.002 1000000 0.0033 0.37 0.025 24.65 0.41 1.6 2.4
Day10 Lane1 EPI_119 5.1 0.0051 15 0.002 1000000 0.0033 0.39 0.026 25.76 0.39 1.6 2.4
Day10 Lane1 EPI_120 4.58 0.0046 15 0.002 1000000 0.0033 0.35 0.023 23.13 0.43 1.7 2.3
Day10 Lane1 EPI_127 4.58 0.0046 15 0.002 1000000 0.0033 0.35 0.023 23.13 0.43 1.7 2.3
Day10 Lane1 EPI_128 4.8 0.0048 15 0.002 1000000 0.0033 0.36 0.024 24.24 0.41 1.7 2.4
Day10 Lane1 EPI_135 4.08 0.0041 15 0.002 1000000 0.0033 0.31 0.021 20.61 0.49 1.9 2.1
Day10 Lane1 EPI_136 3.7 0.0037 15 0.002 1000000 0.0033 0.28 0.019 18.69 0.54 2.1 1.9
Day10 Lane1 EPI_143 2.88 0.0029 15 0.002 1000000 0.0033 0.22 0.015 14.55 0.69 2.8 1.3
Day10 Lane1 EPI_145 2.92 0.0029 15 0.002 1000000 0.0033 0.22 0.015 14.75 0.68 2.7 1.3

Sample QC at Genewiz

20161216

Pool QC Genewiz

Samples passed QC and were confirmed for sequencing on a single lane of Illumina Highseq2500 2x100bp on 20161219

Written on December 1, 2016