SCELLECTOR: ranking amplification bias in single cells using shallow sequencing

Vivekananda Sarangi,Alexej Abyzov,Arijit Panda,Taejeong Bae,Flora Vaccarino,Alexandre Jourdon

doi:10.1186/s12859-020-03858-y

Vivekananda Sarangi, Alexej Abyzov + Show 4 more

Open Access

https://doi.org/10.1186/s12859-020-03858-y

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Journal: BMC Bioinformatics	Publication Date: Nov 12, 2020
Citations: 3	License type: open-access

Affiliation: Mayo Clinic, Yale University

Abstract

BackgroundThe study of mosaic mutation is important since it has been linked to cancer and various disorders. Single cell sequencing has become a powerful tool to study the genome of individual cells for the detection of mosaic mutations. The amount of DNA in a single cell needs to be amplified before sequencing and multiple displacement amplification (MDA) is widely used owing to its low error rate and long fragment length of amplified DNA.However, the phi29 polymerase used in MDA is sensitive to template fragmentation and presence of sites with DNA damage that can lead to biases such as allelic imbalance, uneven coverage and over representation of C to T mutations. It is therefore important to select cells with uniform amplification to decrease false positives and increase sensitivity for mosaic mutation detection.ResultsWe propose a method, Scellector (single cell selector), which uses haplotype information to detect amplification quality in shallow coverage sequencing data. We tested Scellector on single human neuronal cells, obtained in vitro and amplified by MDA. Qualities were estimated from shallow sequencing with coverage as low as 0.3× per cell and then confirmed using 30× deep coverage sequencing. The high concordance between shallow and high coverage data validated the method.ConclusionScellector can potentially be used to rank amplifications obtained from single cell platforms relying on a MDA-like amplification step, such as Chromium Single Cell profiling solution.

Highlights

The study of mosaic mutation is important since it has been linked to cancer and various disorders
Whole genome sequencing of bulk tissue has been used for detecting somatic mutations, it is not sensitive enough to detect mosaic mutations present below 1% variant allele frequency (VAF), i.e., a heterozygous mutation present in less than 2% of the cells
After Whole Genome Amplification (WGA), not all cells are amplified uniformly owing to the allelic imbalance described earlier

Summary

Introduction

The study of mosaic mutation is important since it has been linked to cancer and various disorders. Some somatic mutations might give the cells proliferative advantage, and cause cancer, or Sarangi et al BMC Bioinformatics (2020) 21:521 can affect the cellular functions without a proliferative effect This makes the detection of mosaic mutation important for understanding the mechanism of various diseases. Whole genome sequencing of bulk tissue has been used for detecting somatic mutations, it is not sensitive enough to detect mosaic mutations present below 1% variant allele frequency (VAF), i.e., a heterozygous mutation present in less than 2% of the cells This hurdle has been overcome by single-cell DNA sequencing (scDNA-seq) which in recent times has emerged as an efficient tool for studying mosaic mutations [7,8,9]. MDA is the most widely used method for WGA owing to its longer fragment length (up to 70 kbps), low error rate during amplification and higher fraction of the genome being amplified as compared to the other WGA methods [13]

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