Variant calling pipeline for amplicon-based sequencing of the SARS-Cov-2 viral genome
Overview of the analytic pipeline “Donner” for variant calling from amplicon-based sequencing of the SARS-Cov-2 viral genome sequences
Created date: 2020/5/16
Update date: 2020/11/27
Kenjiro Kosaki,
Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan
Following article has been has published in the Journal of Hospital Infection.
Clinical Utility of SARS-CoV-2 Whole Genome Sequencing in Deciphering Source of Infection
（https://www.sciencedirect.com/science/article/pii/S0195670120304953）
Toshiki Takenouchi, Yuka W. Iwasaki, Sei Harada, Hirotsugu Ishizu, Yoshifumi Uwamino, Shunsuke Uno, Asami Osada, Naoki Hasegawa, Mitsuru Murata, Toru Takebayashi, Koichi Fukunaga, Hideyuki Saya, Yuko Kitagawa, Masayuki Amagai, Haruhiko Siomi, Kenjiro Kosaki
doi: http://dx.doi.org/10.1016/j.jhin.2020.10.014
System requirements
The system was tested on Cent OS 6.3 and Ubuntu 16.04LTS and 18.06LTS.
Conda may be helpful during installation of the required packages
Reference SARS-Cov-2 sequence
MN908947.3.fasta was used as the reference
Wu,F., Zhao,S., Yu,B., Chen,Y.M., Wang,W., Song,Z.G., Hu,Y.,Tao,Z.W., Tian,J.H., Pei,Y.Y., Yuan,M.L., Zhang,Y.L., Dai,F.H.,Liu,Y., Wang,Q.M., Zheng,J.J., Xu,L., Holmes,E.C. and Zhang,Y.Z.
A new coronavirus associated with human respiratory disease in China.
Nature 2020; 579:265-269
Raw data generation
PCR amplified using the ARITC primer set version 3
Amplicon sequencing by Illumina MiSeq
Software components used in the pipeline
Resampling and quality control
seqtk
Resample fastq data when depth is excessively high
Wei Shen, Shuai Le, Yan Li, and Fuquan Hu
SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation
PLoS One. 2016; 11: e0163962.
fastp
Trim adapters
Chen S, Zhou Y, Chen Y, Gu J.
fastp: an ultra-fast all-in-one FASTQ preprocessor.
Bioinformatics. 2018;34:i884-i890.
ivar
Soft clip PCR primer sequences
Grubaugh ND, Gangavarapu K, Quick J, Matteson NL, De Jesus JG, Main BJ, Tan AL, Paul LM, Brackney DE, Grewal S, Gurfield N, Van Rompay KKA, Isern S, Michael SF, Coffey LL, Loman NJ, Andersen KG.
An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar.
Genome Biol. 2019 Jan 8;20:8.
Alignment
bwa version xx or above
Li H, Durbin R.
Fast and accurate short read alignment with Burrows-Wheeler transform.
Bioinformatics. 2009;25:1754-60.
variant calling
samtools (version1.9 or above)
bcftools (version 1.9 or above)
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup.
The Sequence Alignment/Map format and SAMtools.
Bioinformatics. 2009;25:2078-9.
ivar
See above
Annotation
SnpEff
Predicted effects on translated protein
Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM.
A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.
Fly (Austin). 2012 ;6:80-92.
Requires to build custom database for SARS-COV-2
Custom GFF3 file for building database
See atached GFF3 file.
Binary database
covid19
ANNOVAR
Annotate mutations considering the ribosome slippage event in ORF1ab
Wang K, Li M, Hakonarson H. ANNOVAR: Functional annotation of genetic variants from next-generation sequencing data Nucleic Acids Research, 38:e164, 2010
For ANNOVAR installation and how to analyze mutations in SARS-CoV-2, please refer to the ANNOVAR Documentation
(https://doc-openbio.readthedocs.io/projects/annovar/en/latest/).
Validation
water
Pairwise sequence alignment of the sample sequence and the reference sequence base on the Smith-Waterman algorithm.
From the emboss package
Carver T, Bleasby A: The design of Jemboss: a graphical user interface to EMBOSS. Bioinformatics. 2003; 19: 1837-1843.