Whole-genome haplotyping by dilution, amplification, and sequencing
Whole-genome haplotyping by dilution, amplification, and sequencing
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DOI：
10.1073/pnas.1218696110
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发表时间：
2013-04-02
影响因子：
11.1
通讯作者：
Fan, Jian-Bing
中科院分区：
文献类型：
--
作者：
Kaper, Fiona;Swamy, Sajani;Fan, Jian-Bing
标准的全基因组基因分型技术无法确定单倍型。在这里，我们描述了一种快速和经济有效的远程单倍体鉴定方法。基因组DNA被稀释并分配成多个等分，使得每个等分得到单倍体拷贝的一小部分。每个等分中的DNA模板通过多次置换扩增进行扩增，使用NextEra技术转换成条形码测序文库，并在多重池中进行测序。为了评估我们方法的性能，我们将两个男性基因组DNA样本以相等的比例组合，得到一个具有已知单倍型的二倍体X染色体样本。对多重测序文库池进行定向下拉X染色体Duchenne肌营养不良症基因的1-Mb邻接区。我们能够将Duchenne肌营养不良症区域分成两个连续的单倍型块，平均长度为494kb。单倍型与通过测序进行个体DNA一致的碱基调用显示99%的符合率。随后，我们使用该策略对两个人类基因组进行了单倍型分析。用于鉴定样本中所有杂合子SNPs的标准基因组测序与基于稀释扩增的测序数据相结合来解析所鉴定的杂合子SNPs的阶段。使用该程序，我们能够从二倍体序列数据中获得95%的杂合子SNPs。约鲁巴男性DNA的N50为702kb，而欧洲女性DNA的N50为358kb。因此，这里描述的策略适用于一组靶区以及整个基因组的单倍型。
Standard whole-genome genotyping technologies are unable to determine haplotypes. Here we describe a method for rapid and cost-effective long-range haplotyping. Genomic DNA is diluted and distributed into multiple aliquots such that each aliquot receives a fraction of a haploid copy. The DNA template in each aliquot is amplified by multiple displacement amplification, converted into barcoded sequencing libraries using Nextera technology, and sequenced in multiplexed pools. To assess the performance of our method, we combined two male genomic DNA samples at equal ratios, resulting in a sample with diploid X chromosomes with known haplotypes. Pools of the multiplexed sequencing libraries were subjected to targeted pull-down of a 1-Mb contiguous region of the X-chromosome Duchenne muscular dystrophy gene. We were able to phase the Duchenne muscular dystrophy region into two contiguous haplotype blocks with a mean length of 494 kb. The haplotypes showed 99% agreement with the consensus base calls made by sequencing the individual DNAs. We subsequently used the strategy to haplotype two human genomes. Standard genomic sequencing to identify all heterozygous SNPs in the sample was combined with dilution-amplification-based sequencing data to resolve the phase of identified heterozygous SNPs. Using this procedure, we were able to phase >95% of the heterozygous SNPs from the diploid sequence data. The N50 for a Yoruba male DNA was 702 kb whereas the N50 for a European female DNA was 358 kb. Therefore, the strategy described here is suitable for haplotyping of a set of targeted regions as well as of the entire genome.