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A Short‐Read Multiplex Sequencing Method for Reliable, Cost‐Effective and High‐Throughput Genotyping in Large‐Scale Studies
Author(s) -
Cao Hongzhi,
Wang Yu,
Zhang Wei,
Chai Xianghua,
Zhang Xiandong,
Chen Shiping,
Yang Fan,
Zhang Caifen,
Guo Yulai,
Liu Ying,
Tang Zhoubiao,
Chen Caifen,
Xue Yaxin,
Zhen Hefu,
Xu Yinyin,
Rao Bin,
Liu Tao,
Zhao Meiru,
Zhang Wenwei,
Li Yingrui,
Zhang Xiuqing,
Tellier Laurent C. A. M.,
Krogh Anders,
Kristiansen Karsten,
Wang Jun,
Li Jian
Publication year - 2013
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22439
Subject(s) - sanger sequencing , genotyping , biology , amplicon , genetics , computational biology , dna sequencing , typing , throughput , genotype , polymerase chain reaction , gene , computer science , telecommunications , wireless
Accurate genotyping is important for genetic testing. Sanger sequencing‐based typing is the gold standard for genotyping, but it has been underused, due to its high cost and low throughput. In contrast, short‐read sequencing provides inexpensive and high‐throughput sequencing, holding great promise for reaching the goal of cost‐effective and high‐throughput genotyping. However, the short‐read length and the paucity of appropriate genotyping methods, pose a major challenge. Here, we present RCHSBT —reliable, cost‐effective and high‐throughput sequence based typing pipeline—which takes short sequence reads as input, but uses a unique variant calling, haploid sequence assembling algorithm, can accurately genotype with greater effective length per amplicon than even S anger sequencing reads. The RCHSBT method was tested for the human MHC loci HLA‐A , HLA‐B , HLA‐C , HLA‐DQB 1 , and HLA‐DRB 1 , upon 96 samples using Illumina PE 150 reads. Amplicons as long as 950 bp were readily genotyped, achieving 100% typing concordance between RCHSBT ‐called genotypes and genotypes previously called by S anger sequence. Genotyping throughput was increased over 10 times, and cost was reduced over five times, for RCHSBT as compared with S anger sequence genotyping. We thus demonstrate RCHSBT to be a genotyping method comparable to S anger sequencing‐based typing in quality, while being more cost‐effective, and higher throughput.