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Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
14:06

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

Published on: June 23, 2012

Variant identification in multi-sample pools by illumina genome analyzer sequencing.

Rebecca L Margraf1, Jacob D Durtschi, Shale Dames

  • 1ARUP Institute for Clinical & Experimental Pathology®, Salt Lake City, Utah, USA. rebecca.margraf@aruplab.com

Journal of Biomolecular Techniques : JBT
|July 9, 2011
PubMed
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Multi-sample pooling with Illumina sequencing reliably detects genetic variants. This study confirms pooling up to 50 samples is feasible for population sequence variation analysis, aiding molecular diagnostics.

Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • High-throughput sequencing enables population sequence variation analysis.
  • Multi-sample pooling strategies are crucial for cost-effective, large-scale genetic studies.
  • Previous work suggested pooling up to 30 samples for reliable singleton variant detection.

Purpose of the Study:

  • To test the maximum sample pooling limit for reliable singleton variant detection using Illumina Genome Analyzer (GA) sequencing.
  • To evaluate the impact of recent protocol improvements, Illumina GAIIx upgrades, and longer read chemistry on pooling efficiency.
  • To validate variant detection accuracy with pooled samples compared to a control sample.

Main Methods:

  • Utilized multi-sample pooling (30 and 50 samples) with Illumina GAIIx sequencing.
Keywords:
Illumina Genome AnalyzerRETmassively parallel sequencingnext generation sequencingpooling

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  • Employed the SequalPrep(TM) method for amplicon normalization prior to pooling.
  • Analyzed data using variant read percentages and a subtractive correction method with a control sample.

    • Main Results:

    • Detected 59 variants in total, including all 47 known true variants.
    • Singleton variants in 30-sample pools averaged 1.62% reads; in 50-sample pools, 1.01% reads.
    • Longer read lengths (76 bases) exhibited higher error rates, hindering singleton variant distinction.

    Conclusions:

    • Pooling up to 50 samples is reliable for singleton variant detection, contingent on error rates and sequencing coverage.
    • Developed pooling protocols and analysis methods are applicable for variant discovery in diverse genes.
    • Facilitates the design and interpretation of molecular diagnostic tests.