tailieunhanh - Báo cáo y học: "Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries"

Tuyển tập các báo cáo nghiên cứu về y học được đăng trên tạp chí y học Wertheim cung cấp cho các bạn kiến thức về ngành y đề tài: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. | Aird et al. Genome Biology 2011 12 R18 http 2011 12 2 R18 w Genome Biology METHOD Open Access Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries 11 2 2 3 1 Daniel Aird Michael G Ross Wei-Sheng Chen Maxwell Danielsson Timothy Fennell Carsten Russ David B Jaffe 1 Chad Nusbaum1 Andreas Gnirke1 Abstract Despite the ever-increasing output of Illumina sequencing data loci with extreme base compositions are often under-represented or absent. To evaluate sources of base-composition bias we traced genomic sequences ranging from 6 to 90 GC through the process by quantitative PCR. We identified PCR during library preparation as a principal source of bias and optimized the conditions. Our improved protocol significantly reduces amplification bias and minimizes the previously severe effects of PCR instrument and temperature ramp rate. Background The Illumina sequencing platform 1 like other massively parallel sequencing platforms 2 3 continues to produce ever-increasing amounts of data yet suffers from under-representation and reduced quality at loci with extreme base compositions that are recalcitrant to the technology 1 4-6 . Uneven coverage due to base composition necessitates sequencing to excessively high mean coverage for de novo genome assembly 7 and for sensitive polymorphism discovery 8 9 . Although loci with extreme base composition constitute only a small fraction of the human genome they include biologically and medically relevant re-sequencing targets. For example 104 of the first 136 coding bases of the retinoblastoma tumor suppressor gene RB1 are G or C. Traditional Sanger sequencing has long been known to suffer from problems related to the base composition of sequencing templates. GC-rich stretches led to compression artifacts. Polymerase slippage in poly A runs and AT dinucleotide repeats caused mixed sequencing ladders and poor read quality. Processes upstream of the actual sequencing such as cloning introduced