Pushkarev Dmitry, Neff Norma F, Quake Stephen R
Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, California, USA.
Nat Biotechnol. 2009 Sep;27(9):847-50. doi: 10.1038/nbt.1561. Epub 2009 Aug 10.
Recent advances in high-throughput DNA sequencing technologies have enabled order-of-magnitude improvements in both cost and throughput. Here we report the use of single-molecule methods to sequence an individual human genome. We aligned billions of 24- to 70-bp reads (32 bp average) to approximately 90% of the National Center for Biotechnology Information (NCBI) reference genome, with 28x average coverage. Our results were obtained on one sequencing instrument by a single operator with four data collection runs. Single-molecule sequencing enabled analysis of human genomic information without the need for cloning, amplification or ligation. We determined approximately 2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1% as validated by Sanger sequencing and 99.8% concordance with SNP genotyping arrays. We identified 752 regions of copy number variation by analyzing coverage depth alone and validated 27 of these using digital PCR. This milestone should allow widespread application of genome sequencing to many aspects of genetics and human health, including personal genomics.
高通量DNA测序技术的最新进展已在成本和通量方面实现了数量级的提升。在此，我们报告了使用单分子方法对单个人类基因组进行测序的情况。我们将数十亿条24至70碱基对（平均32碱基对）的读段比对到了美国国立生物技术信息中心（NCBI）参考基因组的约90%，平均覆盖度为28倍。我们的结果是由一名操作人员在一台测序仪器上通过四次数据收集运行获得的。单分子测序无需克隆、扩增或连接即可对人类基因组信息进行分析。经桑格测序验证，我们确定了约280万个单核苷酸多态性（SNP），其假阳性率低于1%，且与SNP基因分型阵列的一致性达99.8%。我们仅通过分析覆盖深度就识别出了752个拷贝数变异区域，并使用数字PCR验证了其中27个。这一里程碑事件应能使基因组测序在遗传学和人类健康的诸多方面，包括个人基因组学中得到广泛应用。
