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.
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.
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. 