Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.
Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.
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DOI：
10.1186/1471-2164-13-1
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发表时间：
2012-01-03
期刊：
影响因子：
4.4
通讯作者：
Quail MA
中科院分区：
文献类型：
--
作者：
Oyola SO;Otto TD;Gu Y;Maslen G;Manske M;Campino S;Turner DJ;Macinnis B;Kwiatkowski DP;Swerdlow HP;Quail MA
大规模并行测序技术正在彻底改变基因组和遗传研究的方法。自问世以来，下一代测序（NGS）的规模和效率迅速提高。尽管取得了这一成功，但对碱基组成极其偏倚的基因组或基因组区域进行测序仍然对当前可用的 NGS 平台构成巨大挑战。一些重要病原生物的基因组，例如恶性疟原虫（高 AT 含量）和结核分枝杆菌（高 GC 含量），显示出极端的碱基组成。采用 PCR 扩增的标准文库制备程序已被证明会导致读取覆盖不均匀，特别是在 AT 和 GC 丰富区域中，从而导致基因组组装和变异分析中出现问题。省略 PCR 扩增的替代文库制备方法需要大量起始材料，因此不适合少量 DNA/RNA，例如来自临床分离株的 DNA/RNA。我们开发并优化了适合少量起始材料并耐受极高 AT 含量序列的文库制备程序。我们使用我们的优化条件与标准方法并行，从非临床和临床分离株（含有约 53% 的宿主污染）制备 Illumina 测序文库。通过分析和比较生成的序列数据的质量，我们表明，涉及 PCR 添加剂 (TMAC) 的优化条件可生成扩增文库，提高 AT 丰富区域的覆盖率，并减少对 GC 中性模板的偏差。我们开发了一种稳健且优化的下一代测序文库扩增方法，适用于 AT 极其丰富的基因组。新的扩增条件显着降低了偏差并保留了碱基组成任一极端的复杂性。这一发展将极大地有利于对由于 DNA 起始材料质量低而经常需要扩增的临床样本进行测序。
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. 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). 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. 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 to low mass of DNA starting material.
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BAMVIEW：在参考序列的上下文中查看映射的读取对齐数据。
影响因子：
5.8
作者：
Carver, Tim;Bohme, Ulrike;Otto, Thomas D.;Parkhill, Julian;Berriman, Matthew
通讯作者：
Berriman, Matthew
DOI：
10.1093/bioinformatics/btq269
发表时间：
2010-07-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Otto TD;Sanders M;Berriman M;Newbold C
通讯作者：
Newbold C
DOI：
10.1093/bioinformatics/btp352
发表时间：
2009-08-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Li H;Handsaker B;Wysoker A;Fennell T;Ruan J;Homer N;Marth G;Abecasis G;Durbin R;1000 Genome Project Data Processing Subgroup
通讯作者：
1000 Genome Project Data Processing Subgroup
DOI：
10.1093/bioinformatics/btp324
发表时间：
2009-07-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Li H;Durbin R
通讯作者：
Durbin R
影响因子：
48
作者：
Kozarewa, Iwanka;Ning, Zemin;Quail, Michael A.;Sanders, Mandy J.;Berriman, Matthew;Turner, Daniel J.
通讯作者：
Turner, Daniel J.