Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA.
Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA.
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DOI：
10.1186/s12977-016-0321-6
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发表时间：
2016-12-20
期刊：
影响因子：
3.3
通讯作者：
Coffin JM
中科院分区：
文献类型：
--
作者：
Boltz VF;Rausch J;Shao W;Hattori J;Luke B;Maldarelli F;Mellors JW;Kearney MF;Coffin JM
虽然下一代测序（NGS）提供了前所未有的深入研究病毒群体的潜力，但在文库构建过程中，PCR错误、扩增偏差和重组限制了其在群体测序和非连锁等位基因频率测量中的应用。在这里，我们报告了一种称为超灵敏单基因组测序（uSGS）的方法，用于NGS文库的构建和分析，消除了PCR错误和重组，并产生了与HIV-1单基因组测序测定的“金标准”相同质量的单基因组序列，但深度超过100倍。引物ID标记的cDNA由克隆的BH10野生型和突变型HIV-1转录本的混合物合成，其中包含10个耐药突变。首先，利用uSGS和长PCR引物连接NGS接头（LP-PCR-1）两种方法对所得cDNA进行分离，并平行生成NGS文库。其次，对cDNA进行分割并生成NGS文库，比较3种方法：uSGS和2种采用长PCR引物连接接头的方法（LP-PCR-2和LP-PCR-3）。一致地，uSGS方法扩增的cdna比例更高，平均为30%，而LP-PCR-1为13%，LP-PCR-2为21%，LP-PCR-3为14%。最重要的是，当uSGS序列根据引物id分箱时，94%的分箱不包含PCR重组序列，而LP-PCR-1、2和3分别只有55%、75%和65%。最后，当uSGS应用于HIV-1感染供者的血浆样本时，在每个样本中都检测到频繁和罕见的变异，邻居连接树揭示了由这些突变连锁驱动的基因组簇，表明数据集中缺乏PCR重组。uSGS检测可用于准确检测罕见变异，并确定与HIV-1耐药性相关的罕见等位基因的连锁。此外，该方法可以对体内病毒种群复杂的遗传关系进行准确深入的分析。
Although next generation sequencing (NGS) offers the potential for studying virus populations in unprecedented depth, PCR error, amplification bias and recombination during library construction have limited its use to population sequencing and measurements of unlinked allele frequencies. Here we report a method, termed ultrasensitive Single-Genome Sequencing (uSGS), for NGS library construction and analysis that eliminates PCR errors and recombinants, and generates single-genome sequences of the same quality as the “gold-standard” of HIV-1 single-genome sequencing assay but with more than 100-fold greater depth. Primer ID tagged cDNA was synthesized from mixtures of cloned BH10 wild-type and mutant HIV-1 transcripts containing ten drug resistance mutations. First, the resultant cDNA was divided and NGS libraries were generated in parallel using two methods: uSGS and a method applying long PCR primers to attach the NGS adaptors (LP-PCR-1). Second, cDNA was divided and NGS libraries were generated in parallel comparing 3 methods: uSGS and 2 methods adapted from more recent reports using variations of the long PCR primers to attach the adaptors (LP-PCR-2 and LP-PCR-3). Consistently, the uSGS method amplified a greater proportion of cDNAs, averaging 30% compared to 13% for LP-PCR-1, 21% for LP-PCR-2 and 14% for LP-PCR-3. Most importantly, when the uSGS sequences were binned according to their primer IDs, 94% of the bins did not contain PCR recombinant sequences versus only 55, 75 and 65% for LP-PCR-1, 2 and 3, respectively. Finally, when uSGS was applied to plasma samples from HIV-1 infected donors, both frequent and rare variants were detected in each sample and neighbor-joining trees revealed clusters of genomes driven by the linkage of these mutations, showing the lack of PCR recombinants in the datasets. The uSGS assay can be used for accurate detection of rare variants and for identifying linkage of rare alleles associated with HIV-1 drug resistance. In addition, the method allows accurate in-depth analyses of the complex genetic relationships of viral populations in vivo.
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影响因子：
3.3
作者：
Gall A;Kaye S;Hué S;Bonsall D;Rance R;Baillie GJ;Fidler SJ;Weber JN;McClure MO;Kellam P;SPARTAC Trial Investigators
通讯作者：
SPARTAC Trial Investigators
DOI：
10.1101/gr.1.4.241
发表时间：
1992-05-01
期刊：
PCR methods and applications
影响因子：
--
作者：
Walsh, P S;Erlich, H A;Higuchi, R
通讯作者：
Higuchi, R
影响因子：
3.8
作者：
Palmer, S;Boltz, V;Coffin, JM
通讯作者：
Coffin, JM
影响因子：
14.9
作者：
Casbon JA;Osborne RJ;Brenner S;Lichtenstein CP
通讯作者：
Lichtenstein CP
影响因子：
6.7
作者：
Kearney MF;Spindler J;Shao W;Yu S;Anderson EM;O'Shea A;Rehm C;Poethke C;Kovacs N;Mellors JW;Coffin JM;Maldarelli F
通讯作者：
Maldarelli F