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

• Main Authors: Qiaoling Li, Xia Zhao, Wenwei Zhang, Lin Wang, Jingjing Wang, Dongyang Xu, Zhiying Mei, Qiang Liu, Shiyi Du, Zhanqing Li, Xinming Liang, Xiaman Wang, Hanmin Wei, Pengjuan Liu, Jing Zou, Hanjie Shen, Ao Chen, Snezana Drmanac, Jia Sophie Liu, Li Li, Hui Jiang, Yongwei Zhang, Jian Wang, Huanming Yang, Xun Xu, Radoje Drmanac, Yuan Jiang
• Format: Article
• Language: English
• Published: BMC 2019-03-01
• Series: BMC Genomics
• Subjects: NGS; Multiplex sequencing; Rare index mis-assignment; DNA nanoball technology
• Online Access: http://link.springer.com/article/10.1186/s12864-019-5569-5

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.