---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-01-28 21:54:17+00:00
layout: post
slug: bisuflite-ngs-library-prep-c-gigas-larvae-oa-bisulfite-library-quantification
title: Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite library quantification
categories:
  - 2015
  - Crassostrea gigas larvae OA (2011) bisulfite sequencing
tags:
  - BS-seq
  - Crassostrea gigas
  - DNA Quantification
  - FLx800 plate reader
  - library prep
  - OA
  - ocean acidification
  - Pacific oyster
  - Quant-iT DNA BR Kit
---

The two completed BS Illumina libraries (400ppm and 1000ppm) were quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of  each sample and the standards.  All standards were run in triplicate.  Due to limited sample, the two libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek).



Results:

The standard curve, raw fluorescence, and calculated concentrations (as determined by the Gen5 (BioTek) software) can be seen here: [20150128_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants](https://docs.google.com/spreadsheets/d/1Aw37LJaitJVHMT7E66vC_ChFqGgT2UaCM9WegK5zgtw/edit?usp=sharing)

The standard curve was excellent, exhibiting a R² value = 0.999



<table >
<tbody >
<tr >

<td style="text-align: center;" >**Sample**
</td>

<td style="text-align: center;" >**Concentration (ng/uL)**
</td>
</tr>
<tr >

<td >400ppm
</td>

<td >10.592
</td>
</tr>
<tr >

<td >1000ppm
</td>

<td >0.0
</td>
</tr>
</tbody>
</table>



The 400ppm library looks great, with a good yield.

The 1000ppm library appears to have no measurable quantity of DNA in it.  This is surprising, and disconcerting, as both samples were processed in parallel.  As such, there should be virtually no difference between them, in regards to the library construction process and subsequent yields.

To verify that this wasn't a pipetting error on my part, I re-quantified the 1000ppm library (in duplicate) and still no detectable DNA.

Will repeat the bisulfite conversion and library construction process on the 1000ppm sample in order to generate a usable library for sequencing.