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

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Massively parallel sequencing is affordable, but index mis-assignments are a problem. BGI

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Massively parallel sequencing (MPS) enables affordable genetic testing through sample multiplexing.
  • However, significant index mis-assignments (over 1%) plague some sequencing platforms.
  • This issue necessitates improved methods for accurate sample identification in high-throughput sequencing.

Purpose of the Study:

  • To investigate and address the quality issue of index mis-assignments on BGI sequencers.
  • To evaluate the performance of different library preparation methods in conjunction with BGI's sequencing technology.
  • To assess the sample-to-sample mis-assignment rates using BGI's DNA nanoball (DNB) technology.

Main Methods:

  • Utilized three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR.
  • Employed BGI sequencers featuring DNA nanoball (DNB) technology, which uses rolling circle replication for PCR-free amplification.
  • Quantified single index mis-assignment from free indexed oligos and sample-to-sample mis-assignment rates.

Main Results:

  • Demonstrated single index mis-assignment from free indexed oligos at a rate of 1 in 36 million reads.
  • Showcased virtually no index hopping during DNA nanoball (DNB) creation and arraying.
  • Achieved unprecedentedly low sample-to-sample mis-assignment rates (0.0001% to 0.0004%) with DNB-based NGS libraries under recommended procedures.

Conclusions:

  • Single indexing combined with BGI's DNA nanoball (DNB) technology offers a robust solution to index mis-assignments.
  • This approach provides a simple yet effective method for highly sensitive genetic assays involving numerous samples.
  • The findings support the use of DNB technology for accurate and reliable high-throughput genetic testing.