SEQanswers

Go Back   SEQanswers > Literature Watch



Similar Threads
Thread Thread Starter Forum Replies Last Post
RNA-Seq: High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation. Newsbot! Literature Watch 1 01-21-2013 11:48 PM
PubMed: Large scale library generation for high throughput sequencing. Newsbot! Literature Watch 0 05-19-2011 11:30 PM
RNA-Seq: Method for improved Illumina sequencing library preparation using NuGEN Ovat Newsbot! Literature Watch 0 04-14-2011 02:50 AM
PubMed: Alta-Cyclic: a self-optimizing base caller for next-generation sequencing ECO Literature Watch 1 02-19-2010 07:22 AM
PubMed: Native chromatin preparation and illumina/solexa library construction. Newsbot! Literature Watch 0 02-12-2010 02:12 AM

Reply
 
Thread Tools
Old 01-05-2012, 10:20 AM   #1
Newsbot!
RSS Posting Maniac
 

Join Date: Feb 2008
Posts: 1,443
Default PubMed: Optimizing Illumina Next-Generation Sequencing library preparation for extrem

Syndicated from PubMed RSS Feeds

Optimizing Illumina Next-Generation Sequencing library preparation for extremely AT-biased genomes.

BMC Genomics. 2012 Jan 3;13(1):1

Authors: Oyola SO, Otto TD, Gu Y, Maslen G, Manske M, Campino S, Turner DJ, Maclnnis B, Kwiatkowski DP, Swerdlow HP, Quail MA

Abstract
ABSTRACT: BACKGROUND: Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. RESULTS: We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing 53% host contamination).parasites. By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. CONCLUSION: We have developed a robust and optimized Next-Generation sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due low mass of DNA starting material.


PMID: 22214261 [PubMed - as supplied by publisher]



More...
Newsbot! is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 08:15 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO