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Old 03-16-2009, 07:03 PM   #1
ECO
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Default Sanger publishes amplification-free Illumina library prep paper

Pretty interesting paper from Sanger. Link here.

Looks like they basically add the flow cell primer portion to the standard Y-adapter to enrich directly on the flow cell, eliminating the PCR step and some bias.

GenomeWeb discusses it a bit here.


****************************************************
Nature Methods
Published online: 15 March 2009 | doi:10.1038/nmeth.1311

Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes

Iwanka Kozarewa1,2, Zemin Ning1,2, Michael A Quail1, Mandy J Sanders1, Matthew Berriman1 & Daniel J Turner1

Abstract

Amplification artifacts introduced during library preparation for the Illumina Genome Analyzer increase the likelihood that an appreciable proportion of these sequences will be duplicates and cause an uneven distribution of read coverage across the targeted sequencing regions. As a consequence, these unfavorable features result in difficulties in genome assembly and variation analysis from the short reads, particularly when the sequences are from genomes with base compositions at the extremes of high or low G+C content. Here we present an amplification-free method of library preparation, in which the cluster amplification step, rather than the PCR, enriches for fully ligated template strands, reducing the incidence of duplicate sequences, improving read mapping and single nucleotide polymorphism calling and aiding de novo assembly. We illustrate this by generating and analyzing DNA sequences from extremely (G+C)-poor (Plasmodium falciparum), (G+C)-neutral (Escherichia coli) and (G+C)-rich (Bordetella pertussis) genomes.
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Old 03-17-2009, 02:52 PM   #2
greigite
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Default library quantification

I'm curious how necessary it is to use their qPCR method versus a Bioanalyzer for quantification of the no-PCR libraries. Since I'm just getting started with Illumina sequencing I don't have already sequenced libraries hanging around that I can use as qPCR standards, so the barrier to setting up this method is somewhat high. Could I still get useful results with Bioanalyzer quantification of the no-PCR libraries?
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Old 04-22-2009, 03:06 PM   #3
ewilbanks
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Default

From what I understand, the qPCR step is necessary to make sure you have enough adapters ligated sequence, with both adapters (so it can amplify in the flow cell).

If you just look at total [DNA] I don't think you can tell this, since it will include those sequences with only 1 or no adapters. Maybe you could ask around to find people that have known standards!
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Old 11-02-2009, 01:02 PM   #4
btarrier
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Default qPCR without library standards

Quote:
Originally Posted by greigite View Post
I'm curious how necessary it is to use their qPCR method versus a Bioanalyzer for quantification of the no-PCR libraries. Since I'm just getting started with Illumina sequencing I don't have already sequenced libraries hanging around that I can use as qPCR standards, so the barrier to setting up this method is somewhat high. Could I still get useful results with Bioanalyzer quantification of the no-PCR libraries?

One way to do qPCR without library standards is to use the Illumina supplied PhiX library, which is at a known 10nM and should behave the same from qPCR run to run. Once you make dilutions of this library you can use them for multiple quantifications.
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