Prepping remaining 76 RRBS libraries for geoduck juvenile OA acclimatization study.

20161206

Chose remaining 76 samples from 88 sample set for full timepoint coverage of geoduck ocean acidification study.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

Date	EPI.Tube.Num	Tank	Treatment	TimePoint	Homogenization	DNA.Extraction	DNA.QC	DNA.Conc.ng.µl	DNA.vol.µl	DNA.amount.µg	vol for 100ng	lambda.0.5%	Water.µl	NSBuffer2.µl	MSPI.µl
16-Mar-16	EPI_41	Time0	Field	Day0	20160912	20161025	20161026	13.6	140	1.904	7.4	1	17.6	3	1
16-Mar-16	EPI_42	Time0	Field	Day0	20160912	20161028	20161028	79	140	11.06	1.3	1	23.7	3	1
16-Mar-16	EPI_43	Time0	Field	Day0	20160912	20161025	20161026	17.2	140	2.408	5.8	1	19.2	3	1
16-Mar-16	EPI_44	Time0	Field	Day0	20160912	20161025	20161026	14.8	140	2.072	6.8	1	18.2	3	1
17-Mar-16	EPI_45	Tank11	Low	Day1	20160912	20161028	20161028	43.1	140	6.034	2.3	1	22.7	3	1
17-Mar-16	EPI_46	Tank11	Low	Day1	20160912	20161028	20161028	21.2	140	2.968	4.7	1	20.3	3	1
17-Mar-16	EPI_47	Tank12	Super.Low	Day1	20160912	20161028	20161028	57.6	140	8.064	1.7	1	23.3	3	1
17-Mar-16	EPI_48	Tank12	Super.Low	Day1	20160912	20161028	20161028	12.6	140	1.764	7.9	1	17.1	3	1
17-Mar-16	EPI_49	Tank13	Ambient	Day1	20160912	20161025	20161026	63.4	140	8.876	1.6	1	23.4	3	1
17-Mar-16	EPI_50	Tank13	Ambient	Day1	20160912	20161028	20161028	8.21	140	1.1494	12.2	1	12.8	3	1
17-Mar-16	EPI_51	Tank14	Low	Day1	20160912	20161025	20161026	14.6	140	2.044	6.8	1	18.2	3	1
17-Mar-16	EPI_52	Tank14	Low	Day1	20160912	20161025	20161026	9.82	140	1.3748	10.2	1	14.8	3	1
17-Mar-16	EPI_53	Tank15	Ambient	Day1	20160912	20161025	20161026	27.4	140	3.836	3.6	1	21.4	3	1
17-Mar-16	EPI_54	Tank15	Ambient	Day1	20160912	20161025	20161026	15	140	2.1	6.7	1	18.3	3	1
17-Mar-16	EPI_55	Tank16	Super.Low	Day1	20160912	20161025	20161026	12.4	140	1.736	8.1	1	16.9	3	1
17-Mar-16	EPI_56	Tank16	Super.Low	Day1	20160912	20161025	20161026	21.4	140	2.996	4.7	1	20.3	3	1
29-Jul-16	EPI_151	Ambient	Ambient	Day135	20160909	20161004	20161006	11.8	140	1.652	8.5	1	16.5	3	1
29-Jul-16	EPI_152	Ambient	Ambient	Day135	20160909	20161004	20161006	16.8	140	2.352	6	1	19	3	1
29-Jul-16	EPI_153	Ambient	Ambient	Day135	20160909	20161025	20161026	22.4	140	3.136	4.5	1	20.5	3	1
29-Jul-16	EPI_154	Ambient	Ambient	Day135	20160909	20161025	20161026	19.5	140	2.73	5.1	1	19.9	3	1
29-Jul-16	EPI_159	Low	Low	Day135	20160909	20161004	20161006	21.4	140	2.996	4.7	1	20.3	3	1
29-Jul-16	EPI_160	Low	Low	Day135	20160909	20161004	20161006	10.8	140	1.512	9.3	1	15.7	3	1
29-Jul-16	EPI_161	Low	Low	Day135	20160909	20161028	20161028	24.9	140	3.486	4	1	21	3	1
29-Jul-16	EPI_162	Low	Low	Day135	20160909	20161007	20161010	12.5	140	1.75	8	1	17	3	1
29-Jul-16	EPI_167	Super.Low	Super.Low	Day135	20160908	20160930	Pass	13.2	190	2.508	7.6	1	17.4	3	1
29-Jul-16	EPI_168	Super.Low	Super.Low	Day135	20160908	20160930	Pass	6.18	190	1.1742	16.2	1	8.8	3	1
29-Jul-16	EPI_169	Super.Low	Super.Low	Day135	20160908	20161007	20161010	14.8	140	2.072	6.8	1	18.2	3	1
29-Jul-16	EPI_170	Super.Low	Super.Low	Day135	20160908	20161007	20161010	11.2	140	1.568	8.9	1	16.1	3	1
8-Aug-16	EPI_175	Bin1	Low-Ambient	Day145	20160908	20160930	Pass	19.6	190	3.724	5.1	1	19.9	3	1
8-Aug-16	EPI_176	Bin1	Low-Ambient	Day145	20160908	20160930	Pass	7.84	190	1.4896	12.8	1	12.2	3	1
8-Aug-16	EPI_181	Bin2	Ambient-Ambient	Day145	20160908	20161004	20161006	40.8	140	5.712	2.5	1	22.5	3	1
8-Aug-16	EPI_182	Bin2	Ambient-Ambient	Day145	20160908	20161004	20161006	20.8	140	2.912	4.8	1	20.2	3	1
8-Aug-16	EPI_184	Bin3	Ambient-Ambient	Day145	20160908	20161007	20161010	7.46	140	1.0444	13.4	1	11.6	3	1
8-Aug-16	EPI_185	Bin3	Ambient-Ambient	Day145	20160907	20161007	20161010	18.6	140	2.604	5.4	1	19.6	3	1
8-Aug-16	EPI_187	Bin4	Super.Low-Ambient	Day145	20160907	20160930	Pass	17.1	190	3.249	5.8	1	19.2	3	1
8-Aug-16	EPI_188	Bin4	Super.Low-Ambient	Day145	20160907	20160930	Pass	14.5	190	2.755	6.9	1	18.1	3	1
8-Aug-16	EPI_193	Bin5	Low-Ambient	Day145	20160907	20161004	20161006	9.26	140	1.2964	10.8	1	14.2	3	1
8-Aug-16	EPI_194	Bin5	Low-Ambient	Day145	20160907	20161004	20161006	11.7	140	1.638	8.5	1	16.5	3	1
8-Aug-16	EPI_199	Bin6	Super.Low-Ambient	Day145	20160907	20161004	20161006	21.8	140	3.052	4.6	1	20.4	3	1
8-Aug-16	EPI_200	Bin6	Super.Low-Ambient	Day145	20160907	20161004	20161006	21.2	140	2.968	4.7	1	20.3	3	1
8-Aug-16	EPI_205	Bin7	Ambient-Low	Day145	20160907	20160930	Pass	11.9	190	2.261	8.4	1	16.6	3	1
8-Aug-16	EPI_206	Bin7	Ambient-Low	Day145	20160906	20160930	Pass	18.1	190	3.439	5.5	1	19.5	3	1
8-Aug-16	EPI_208	Bin8	Low-Low	Day145	20160906	20161007	20161010	31	140	4.34	3.2	1	21.8	3	1
8-Aug-16	EPI_209	Bin8	Low-Low	Day145	20160906	20161007	20161010	22	140	3.08	4.5	1	20.5	3	1
8-Aug-16	EPI_214	Bin9	Super.Low-Low	Day145	20160906	20161004	20161006	13.3	140	1.862	7.5	1	17.5	3	1
8-Aug-16	EPI_215	Bin9	Super.Low-Low	Day145	20160906	20161004	20161006	22.6	140	3.164	4.4	1	20.6	3	1
8-Aug-16	EPI_220	Bin10	Super.Low-Low	Day145	20160906	20160930	Pass	48.8	190	9.272	2	1	23	3	1
8-Aug-16	EPI_221	Bin10	Super.Low-Low	Day145	20160906	20160930	Pass	30	190	5.7	3.3	1	21.7	3	1
8-Aug-16	EPI_226	Bin11	Ambient-Low	Day145	20160902	20161007	20161010	13.1	140	1.834	7.6	1	17.4	3	1
8-Aug-16	EPI_227	Bin11	Ambient-Low	Day145	20160902	20161007	20161010	31.6	140	4.424	3.2	1	21.8	3	1
8-Aug-16	EPI_229	Bin12	Low-Low	Day145	20160902	20160930	Pass	17.4	190	3.306	5.7	1	19.3	3	1
8-Aug-16	EPI_230	Bin12	Low-Low	Day145	20160902	20160930	Pass	10.1	190	1.919	9.9	1	15.1	3	1
21-Aug-16	EPI_235	Bin1	Low-Ambient	Day158	20160902	20161004	20161006	10.6	140	1.484	9.4	1	15.6	3	1
21-Aug-16	EPI_236	Bin1	Low-Ambient	Day158	20160902	20161004	20161006	8.66	140	1.2124	11.5	1	13.5	3	1
21-Aug-16	EPI_242	Bin2	Ambient-Ambient	Day158	20160902	20161028	20161028	15.5	140	2.17	6.5	1	18.5	3	1
21-Aug-16	EPI_243	Bin2	Ambient-Ambient	Day158	20160902	20161028	20161028	17.2	140	2.408	5.8	1	19.2	3	1
21-Aug-16	EPI_246	Bin3	Ambient-Ambient	Day158	20160902	20161025	20161026	19.4	140	2.716	5.2	1	19.8	3	1
21-Aug-16	EPI_247	Bin3	Ambient-Ambient	Day158	20160902	20161028	20161028	8.23	140	1.1522	12.2	1	12.8	3	1
21-Aug-16	EPI_250	Bin4	Super.Low-Ambient	Day158	20160901	20161025	20161026	28.2	140	3.948	3.5	1	21.5	3	1
21-Aug-16	EPI_251	Bin4	Super.Low-Ambient	Day158	20160901	20161025	20161026	16	140	2.24	6.3	1	18.7	3	1
21-Aug-16	EPI_257	Bin5	Low-Ambient	Day158	20160901	20161004	20161006	15.4	140	2.156	6.5	1	18.5	3	1
21-Aug-16	EPI_258	Bin5	Low-Ambient	Day158	20160901	20161028	20161028	18.8	140	2.632	5.3	1	19.7	3	1
21-Aug-16	EPI_264	Bin6	Super.Low-Ambient	Day158	20160901	20161025	20161026	8.42	140	1.1788	11.9	1	13.1	3	1
21-Aug-16	EPI_265	Bin6	Super.Low-Ambient	Day158	20160901	20161025	20161026	17.5	140	2.45	5.7	1	19.3	3	1
21-Aug-16	EPI_271	Bin7	Ambient-Low	Day158	20160901	20161028	20161028	39.1	140	5.474	2.6	1	22.4	3	1
21-Aug-16	EPI_272	Bin7	Ambient-Low	Day158	20160901	20161028	20161028	105	140	14.7	1	1	24	3	1
21-Aug-16	EPI_275	Bin8	Low-Low	Day158	20160825	20161025	20161026	15	140	2.1	6.7	1	18.3	3	1
21-Aug-16	EPI_276	Bin8	Low-Low	Day158	20160824	20161025	20161026	21	140	2.94	4.8	1	20.2	3	1
21-Aug-16	EPI_282	Bin9	Super.Low-Low	Day158	20160824	20161004	20161006	10.3	140	1.442	9.7	1	15.3	3	1
21-Aug-16	EPI_283	Bin9	Super.Low-Low	Day158	20160824	20161028	20161028	36.6	140	0.77	2.7	1	22.3	3	1
21-Aug-16	EPI_289	Bin10	Super.Low-Low	Day158	20160824	20161025	20161026	13.4	140	1.876	7.5	1	17.5	3	1
21-Aug-16	EPI_290	Bin10	Super.Low-Low	Day158	20160824	20161025	20161026	7.4	140	1.036	13.5	1	11.5	3	1
21-Aug-16	EPI_298	Bin11	Ambient-Low	Day158	20160824	20161028	20161028	79.9	140	11.186	1.3	1	23.7	3	1
21-Aug-16	EPI_299	Bin11	Ambient-Low	Day158	20160824	20161007	20161010	17.6	140	2.464	5.7	1	19.3	3	1
21-Aug-16	EPI_302	Bin12	Low-Low	Day158	20160824	20161004	20161006	32.8	140	4.592	3	1	22	3	1
21-Aug-16	EPI_303	Bin12	Low-Low	Day158	20160824	20161004	20161006	32.4	140	4.536	3.1	1	21.9	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

• Removed from incubator at 09:45 on 20161207
• Proceeded with EZ DNA Methylation-Gold Kit (Catalog Nos. D5005 & D5006) EZ DNA Methylation-Gold Manual

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

Bisulfite Conversion ssDNA QC

• 1µl was used to assess fragment size on the bioanalyzer.

Expected DNA size for Gold kit (A)

Results of bisulfite converted samples

20161209

• Samples all look good except EPI-44. Will run again

• Samples 52, 53, 54, 55, 56 are as expected, samples 151, 152, 153, 154, 159, 160 are shifted to a larger proportion of smaller fragments

• Samples are shifted to a larger proportion of smaller fragments

20161211

• Samples 185, 188, 193, 200, 205, 206, 208, and 209 are shifted to a larger proportion of smaller fragments

• Samples 214, 229, 230, 235, 236, 242 are shifted to a larger proportion of smaller fragments

• Samples 243, 246, 247, 250, 251, 258, 264, 265, and 272 are shifted to a larger proportion of smaller fragments

• Sample 275 has abnormal peak around 3000
• Sample 44 was re-reun and still has strange very small fragment size
• Samples 275, 276, 282, 283, 298 are shifted to a larger proportion of smaller fragments

20161213

Step 3 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 20161213
• Library prep was finished 20161213
• 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_151	1	ATCACG
EPI_152	2	CGATGT
EPI_153	3	TTAGGC
EPI_154	4	TGACCA
EPI_159	5	ACAGTG
EPI_160	6	GCCAAT
EPI_161	7	CAGATC
EPI_162	8	ACTTGA
EPI_167	9	GATCAG
EPI_168	10	TAGCTT
EPI_169	11	GGCTAC
EPI_170	12	CTTGTA
EPI_175	1	ATCACG
EPI_176	2	CGATGT
EPI_181	3	TTAGGC
EPI_182	4	TGACCA
EPI_184	5	ACAGTG
EPI_185	6	GCCAAT
EPI_187	7	CAGATC
EPI_188	8	ACTTGA
EPI_193	9	GATCAG
EPI_194	10	TAGCTT
EPI_199	11	GGCTAC
EPI_200	12	CTTGTA
EPI_205	1	ATCACG
EPI_206	2	CGATGT
EPI_208	3	TTAGGC
EPI_209	4	TGACCA
EPI_214	5	ACAGTG
EPI_215	6	GCCAAT
EPI_220	7	CAGATC
EPI_221	8	ACTTGA
EPI_226	9	GATCAG
EPI_227	10	TAGCTT
EPI_229	11	GGCTAC
EPI_230	12	CTTGTA
EPI_41	1	ATCACG
EPI_42	2	CGATGT
EPI_43	4	TGACCA

Library Quantification

20161213 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)

• Illumina indicates samples should be >3ng/µl

Sample.ID	ng/µl
EPI_151	0.242
EPI_152	0.486
EPI_153	0.580
EPI_154	0.320
EPI_159	0.356
EPI_160	0.210
EPI_161	0.226
EPI_162	0.930
EPI_167	0.808
EPI_168	0.880
EPI_169	0.500
EPI_170	0.866
EPI_175	0.552
EPI_176	0.676
EPI_181	1.18
EPI_182	0.902
EPI_184	0.956
EPI_185	0.972
EPI_187	0.976
EPI_188	1.05
EPI_193	1.74
EPI_194	0.518
EPI_199	0.464
EPI_200	0.664
EPI_205	0.312
EPI_206	0.270
EPI_208	0.408
EPI_209	0.114
EPI_214	0.350
EPI_215	1.192
EPI_220	0.888
EPI_221	0.492
EPI_226	1.71
EPI_227	0.796
EPI_229	0.808
EPI_230	0.642
EPI_41	0.314
EPI_42	0.740
EPI_43	0.252

• Library concentration is significantly lower than expected in the majority of the samples

Library Quality

20161214

• 1.0µl of each sample was run on the Agilent DNA High Sensitivity Kit

Illumina DNA Methylation kit expected library size on high sensitivity chip

RRBS Chip 1 Data

Conclusions

• Library quality is shifted to a smaller size in libraries that are detectable.
• Given the low concentraiton and poor traces, no further chips were run and the library preps need to be redone.