Format

Send to

Choose Destination
Genome Biol. 2018 Mar 15;19(1):33. doi: 10.1186/s13059-018-1408-2.

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

Author information

1
Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK.
2
Present address: MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, EH4 2XU, UK.
3
Bioinformatics Group, The Babraham Institute, Cambridge, CB22 3AT, UK.
4
Mass Spectrometry Facility, The Babraham Institute, Cambridge, CB22 3AT, UK.
5
Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK. m.branco@qmul.ac.uk.
6
Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK. wolf.reik@babraham.ac.uk.
7
Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK. wolf.reik@babraham.ac.uk.
8
Centre for Trophoblast Research, University of Cambridge, Cambridge, CB2 3EG, UK. wolf.reik@babraham.ac.uk.

Abstract

BACKGROUND:

Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.

RESULTS:

We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies.

CONCLUSIONS:

We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

KEYWORDS:

Artefacts; Biases; Bisulfite conversion; DNA methylation; Degradation; GC skew; NGS; Polymerase; WGBS

PMID:
29544553
PMCID:
PMC5856372
DOI:
10.1186/s13059-018-1408-2
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for BioMed Central Icon for PubMed Central
Loading ...
Support Center