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Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
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Finding a suitable library size to call variants in RNA-Seq.

Anna Quaglieri1,2, Christoffer Flensburg3, Terence P Speed3,4,5

  • 1Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052, Australia. quaglieri.a@wehi.edu.au.

BMC Bioinformatics
|December 2, 2020
PubMed
Summary

For acute myeloid leukemia studies, 30-40 million 100 bp paired-end reads are recommended for optimal somatic mutation detection via RNA sequencing. This library size ensures 90-95% variant recovery, balancing cost and data quality.

Keywords:
Cancer RNA-SeqLibrary sizeSequencing depthVariant calling

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Area of Science:

  • Genomics
  • Bioinformatics
  • Cancer Biology

Background:

  • RNA sequencing (RNA-seq) is crucial for understanding cancer biology by analyzing gene expression and mutations.
  • Library size is a critical factor in RNA-seq studies, impacting cost and data quality.
  • This study investigates the effect of library size on somatic mutation detection in acute myeloid leukemia (AML).

Purpose of the Study:

  • To determine the optimal library size for somatic mutation detection in RNA-seq data.
  • To evaluate the sensitivity of different mutation callers and filtering strategies at varying sequencing depths.
  • To provide recommendations for library preparation in large-scale cancer genomics projects.

Main Methods:

  • Downsampling of high-depth RNA-seq data from 45 AML samples (Leucegene project).
  • Simulation of various library sizes (80M, 50M, 40M, 30M, 20M fragments).
  • Comparison of mutation detection sensitivity using six methods (MuTect, VarScan, VarDict with different filters) on simulated datasets and TCGA-LAML cohort.

Main Results:

  • Sensitivity for detecting somatic mutations decreased with library sizes below 30-40 million fragments.
  • VarDict showed the highest sensitivity for insertions and deletions (60%).
  • Single nucleotide variant sensitivity was consistent across most callers, though MuTect required filter adjustments for RNA-seq data.

Conclusions:

  • A minimum of 30-40 million 100 bp paired-end reads are necessary for 90-95% recovery of variants in recurrently mutated myeloid genes.
  • These findings aid in optimizing RNA-seq experimental design for cost-effective and high-quality cancer mutation detection.
  • Further studies are recommended to explore mutation and gene expression patterns in other cancer types.