Sam's Notebook » NF_18

Sample Submission – BS-seq Library Pool to Genewiz

kubu4 — Tue, 22 Dec 2015 23:00:49 +0000

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.

Illumina Methylation Library Quantification – BS-seq Oly/C.gigas Libraries

kubu4 — Tue, 22 Dec 2015 22:51:34 +0000

0000-0002-2747-368X

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.

Illumina Methylation Library Construction – Oly/C.gigas Bisulfite-treated DNA

kubu4 — Tue, 22 Dec 2015 05:55:42 +0000

0000-0002-2747-368X

Took the bisulfite-treated DNA from 20151218 and made Illumina libraries using the TruSeq DNA Methylation Library Kit (Illumina).

Quantified the completed libraries using the Qubit 3.0 dsDNA BR Kit (ThermoFisher).

Evaluated the DNA with the Bioanalyzer 2100 (Agilent) using the DNA 12000 assay. Illumina recommended using the High Sensitivity assay, but we don’t have access to that so I figured I’d just give the DNA 12000 assay a go.

SampleName	IndexNumber	BarCode
1NF11	1	ATCACG
1NF15	2	CGATGT
1NF16	3	TTAGGC
1NF17	4	TGACCA
2NF5	5	ACAGTG
2NF6	6	GCCAAT
2NF7	7	CAGATC
2NF8	8	ACTTGA
M2	9	GATCAG
M3	10	TAGCTT
NF2_6	11	GGCTAC
NF_18	12	CTTGTA

Results:

Library Quantification (Google Sheet): 20151221_quantification_illumina_methylation_libraries

Test Name	Concentration (ng/μL)
1NF11	Out of range
1NF15	2.14
1NF16	2.74
1NF17	2.64
2NF5	2.92
2NF6	Out of range
2NF7	2.42
2NF8	2.56
M2	Out of range
M3	2.1
NF2_6	2.38
NF2_18	Out of range

I used the Qubit’s BR (broad range) kit because I wasn’t sure what concentrations to expect. I need to use the high sensitivity kit to get a better evaluation of all the samples’ concentrations.

Bioanalyzer Data File (Bioanalyzer 2100): 2100_20expert_DNA_2012000_DE72902486_2015-12-21_16-58-43.xad

Ha! Well, looks like you definitely need to use the DNA High Sensitivty assay for the Bioanalyzer to pick up anything. Although, I guess you can see a slight hump in most of the samples at the appropriate sizes (~300bp); you just have to squint.