Rxivist logo

Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform

By 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

Posted 11 Jun 2018
bioRxiv DOI: 10.1101/343137 (published DOI: 10.1186/s12864-019-5569-5)

Accurate next generation sequencing (NGS) is critical for understanding genetic predisposition to human disease and thus aiding clinical diagnosis and personalized precision medicine. Recent breakthroughs in massively parallel sequencing, especially when coupled with sample multiplexing, have driven sequencing cost down and made clinical genetic tests broadly affordable. However, intractable index mis-assignment (commonly exceeds 1%) has been reported on some widely used sequencing platforms. Burdensome unique dual indexing is now used to reduce this problem. Here, we investigated this quality issue on BGI sequencers using three major library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGI sequencers utilize a unique DNA nanoball (DNB) technology that is based on rolling circle replication (RCR) for array preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrate here that single index mis-assignment from free indexed oligos on these sequencers occurs at a rate of only one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying, as expected for the RCR process. Furthermore, the DNB-based NGS applications have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% using only single indexing. Therefore, single indexing with DNB sequencing technology provides a simple but effective method for sensitive research and clinical genetic assays that require the detection of low abundance sequences in a large number of samples.

Download data

  • Downloaded 1,642 times
  • Download rankings, all-time:
    • Site-wide: 5,870 out of 101,046
    • In genomics: 932 out of 6,266
  • Year to date:
    • Site-wide: 23,967 out of 101,046
  • Since beginning of last month:
    • Site-wide: 28,096 out of 101,046

Altmetric data

Downloads over time

Distribution of downloads per paper, site-wide


Sign up for the Rxivist weekly newsletter! (Click here for more details.)


  • 20 Oct 2020: Support for sorting preprints using Twitter activity has been removed, at least temporarily, until a new source of social media activity data becomes available.
  • 18 Dec 2019: We're pleased to announce PanLingua, a new tool that enables you to search for machine-translated bioRxiv preprints using more than 100 different languages.
  • 21 May 2019: PLOS Biology has published a community page about Rxivist.org and its design.
  • 10 May 2019: The paper analyzing the Rxivist dataset has been published at eLife.
  • 1 Mar 2019: We now have summary statistics about bioRxiv downloads and submissions.
  • 8 Feb 2019: Data from Altmetric is now available on the Rxivist details page for every preprint. Look for the "donut" under the download metrics.
  • 30 Jan 2019: preLights has featured the Rxivist preprint and written about our findings.
  • 22 Jan 2019: Nature just published an article about Rxivist and our data.
  • 13 Jan 2019: The Rxivist preprint is live!