Reliable multiplex sequencing with rare index mis-assignment on DNB-based NGS platform

Massively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms. Here, we investigated this quality issue on BGI sequencers u...

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Bibliographic Details
Published in:BMC genomics 2019-03, Vol.20 (1), p.215-215, Article 215
Main Authors: Li, Qiaoling, Zhao, Xia, Zhang, Wenwei, Wang, Lin, Wang, Jingjing, Xu, Dongyang, Mei, Zhiying, Liu, Qiang, Du, Shiyi, Li, Zhanqing, Liang, Xinming, Wang, Xiaman, Wei, Hanmin, Liu, Pengjuan, Zou, Jing, Shen, Hanjie, Chen, Ao, Drmanac, Snezana, Liu, Jia Sophie, Li, Li, Jiang, Hui, Zhang, Yongwei, Wang, Jian, Yang, Huanming, Xu, Xun, Drmanac, Radoje, Jiang, Yuan
Format: Article
Language:English
Subjects:
Deoxyribonucleic acid
DNA
DNA biosynthesis
DNA nanoball technology
DNA sequencing
Gene sequencing
Genetic research
Genetic testing
Genomes
Genomics
Libraries
Methods
Multiplex sequencing
Multiplexing
NGS
Nucleotide sequence
Polymerase chain reaction
Quality control
Rare index mis-assignment
Technology
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Summary:Massively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms. Here, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGI's sequencers utilize a unique DNA nanoball (DNB) technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001 to 0.0004% under recommended procedures. Single indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.
ISSN:1471-2164
1471-2164
DOI:10.1186/s12864-019-5569-5