---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-12-22 05:55:42+00:00
layout: post
slug: illumina-methylation-library-construction-olyc-gigas-bisulfite-treated-dna
title: Illumina Methylation Library Construction - Oly/C.gigas Bisulfite-treated DNA
categories:
  - 2015
  - BS-seq Libraries for Sequencing at Genewiz
  - Olympia oyster reciprocal transplant
tags:
  - 1NF11
  - 1NF15
  - 1NF16
  - 1NF17
  - 2NF5
  - 2NF6
  - 2NF7
  - 2NF8
  - barcodes
  - bioanalyzer
  - BS-seq
  - Crassostrea gigas
  - DNA 12000 assay
  - electropherogram
  - M2
  - M3
  - NF2_6
  - NF_18
  - olympia oyster
  - Ostrea lurida
  - Pacific oyster
  - Qubit 3.0
  - Qubit dsDNA BR
  - TruSeq
  - TruSeq DNA Methylation Library
---

Took the [bisulfite-treated DNA from 20151218](https://robertslab.github.io/sams-notebook/posts/2015/2015-12-18-bisulfite-treatment-oly-reciprocal-transplant-dna-c-gigas-lotterhos-dna-for-bs-seq/) and made Illumina libraries using the [TruSeq DNA Methylation Library Kit (Illumina)(https://github.com/sr320/LabDocs/blob/master/protocols/Commercial_Protocols/Illumina_truseq-dna-methylation-library-prep-guide-15066014-a.pdf).

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.

<table cellspacing="0" border="0" >
<tbody >
<tr >

<td align="left" height="17" valign="bottom" >**SampleName**
</td>

<td align="left" valign="bottom" >**IndexNumber**
</td>

<td align="left" valign="bottom" >**BarCode**
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >1NF11
</td>

<td align="right" valign="bottom" >1
</td>

<td align="left" valign="bottom" >ATCACG
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >1NF15
</td>

<td align="right" valign="bottom" >2
</td>

<td align="left" valign="bottom" >CGATGT
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >1NF16
</td>

<td align="right" valign="bottom" >3
</td>

<td align="left" valign="bottom" >TTAGGC
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >1NF17
</td>

<td align="right" valign="bottom" >4
</td>

<td align="left" valign="bottom" >TGACCA
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >2NF5
</td>

<td align="right" valign="bottom" >5
</td>

<td align="left" valign="bottom" >ACAGTG
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >2NF6
</td>

<td align="right" valign="bottom" >6
</td>

<td align="left" valign="bottom" >GCCAAT
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >2NF7
</td>

<td align="right" valign="bottom" >7
</td>

<td align="left" valign="bottom" >CAGATC
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >2NF8
</td>

<td align="right" valign="bottom" >8
</td>

<td align="left" valign="bottom" >ACTTGA
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >M2
</td>

<td align="right" valign="bottom" >9
</td>

<td align="left" valign="bottom" >GATCAG
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >M3
</td>

<td align="right" valign="bottom" >10
</td>

<td align="left" valign="bottom" >TAGCTT
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >NF2_6
</td>

<td align="right" valign="bottom" >11
</td>

<td align="left" valign="bottom" >GGCTAC
</td>
</tr>
<tr >

<td align="left" height="17" valign="bottom" >NF_18
</td>

<td align="right" valign="bottom" >12
</td>

<td align="left" valign="bottom" >CTTGTA
</td>
</tr>
</tbody>
</table>



Results:

Library Quantification (Google Sheet): [20151221_quantification_illumina_methylation_libraries](https://docs.google.com/spreadsheets/d/1XGu_J4gJ3ddhjTvEvwoFLUCNe5DQhdvRf1tVxqud--Q/edit?usp=sharing)

<table cellpadding="0" cellspacing="0" border="0" dir="ltr" > 
<tbody >
<tr >

<td data-sheets-value="[null,2,"Test Name"]" >**Test Name**
</td>

<td data-sheets-value="[null,2,"Original sample conc."]" >**Concentration (ng/μL)
**
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF11"]" >1NF11
</td>

<td data-sheets-value="[null,2,"Out of range"]" >Out of range
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF15"]" >1NF15
</td>

<td data-sheets-value="[null,3,null,2.14]" >2.14
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF16"]" >1NF16
</td>

<td data-sheets-value="[null,3,null,2.74]" >2.74
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"1NF17"]" >1NF17
</td>

<td data-sheets-value="[null,3,null,2.64]" >2.64
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF5"]" >2NF5
</td>

<td data-sheets-value="[null,3,null,2.92]" >2.92
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF6"]" >2NF6
</td>

<td data-sheets-value="[null,2,"Out of range"]" >Out of range
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF7"]" >2NF7
</td>

<td data-sheets-value="[null,3,null,2.42]" >2.42
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"2NF8"]" >2NF8
</td>

<td data-sheets-value="[null,3,null,2.56]" >2.56
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"M2"]" >M2
</td>

<td data-sheets-value="[null,2,"Out of range"]" >Out of range
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"M3"]" >M3
</td>

<td data-sheets-value="[null,3,null,2.1]" >2.1
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"NF2_6"]" >NF2_6
</td>

<td data-sheets-value="[null,3,null,2.38]" >2.38
</td>
</tr>
<tr >

<td data-sheets-value="[null,2,"NF2_18"]" >NF2_18
</td>

<td data-sheets-value="[null,2,"Out of range"]" >Out of range
</td>
</tr>
</tbody>
</table>



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](https://eagle.fish.washington.edu/Arabidopsis/Bioanalyzer%20Data/2100%20expert_DNA%2012000_DE72902486_2015-12-21_16-58-43.xad)



![](https://eagle.fish.washington.edu/Arabidopsis/20151221_bioanalyzer_illumina_methylation_libraries.jpg)(http://eagle.fish.washington.edu/Arabidopsis/20151221_bioanalyzer_illumina_methylation_libraries.jpg)

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. ;)