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BMC Genomics. 2019 Mar 13;20(1):215. doi: 10.1186/s12864-019-5569-5.

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

Li Q1,2,3, Zhao X1,2,3, Zhang W1,2,4, Wang L5, Wang J1,2, Xu D1,2, Mei Z3, Liu Q6, Du S3, Li Z1,2,3, Liang X3, Wang X6, Wei H3, Liu P1,2,3, Zou J3, Shen H1,2,3, Chen A1,2, Drmanac S1,5, Liu JS5, Li L1,2, Jiang H3, Zhang Y1,5, Wang J1,7, Yang H1,7, Xu X1,2, Drmanac R8,9,10,11, Jiang Y12.

Author information

1
BGI-Shenzhen, Shenzhen, 518083, China.
2
China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China.
3
MGI, BGI-Shenzhen, Shenzhen, 518083, China.
4
Guangdong High-throughput Sequencing Research Center, Shenzhen, China.
5
Complete Genomics Inc., 2904 Orchard Pkwy, San Jose, California, 95134, USA.
6
BGI Genomics, BGI-Shenzhen, Shenzhen, 518083, China.
7
James D. Watson Institute of Genome Sciences, Hangzhou, 310058, China.
8
BGI-Shenzhen, Shenzhen, 518083, China. rdrmanac@completegenomics.com.
9
China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China. rdrmanac@completegenomics.com.
10
Complete Genomics Inc., 2904 Orchard Pkwy, San Jose, California, 95134, USA. rdrmanac@completegenomics.com.
11
MGI, BGI-Shenzhen, Shenzhen, 518083, China. rdrmanac@completegenomics.com.
12
Complete Genomics Inc., 2904 Orchard Pkwy, San Jose, California, 95134, USA. yjiang@completegenomics.com.

Abstract

BACKGROUND:

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.

RESULTS:

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.

CONCLUSIONS:

Single indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

KEYWORDS:

DNA nanoball technology; Multiplex sequencing; NGS; Rare index mis-assignment

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