Reference genome mapping and variant calling

Elizabeth Permina
16 Mar 2018

##

  • M.tuberculosis whole genome sequence analysis
  • reference genome mapping and variant calling

- Overview:

- Whole genome sequence of 20 samples of MT has been received from the Massey genomic sequencing facility. After initial quality control the reference assembly and variant calling has been done using HR37v MT genome from Genbank DB. The variant calling produced an even amount of SNPs for 17 out of 20 samples with 3 samples producing more than expected for this type of study (Samples 2, 9 and 15). Samples 2, 14 and 15 proved to more related in terms of SNP sets.

tools used

  • Reference mapping: BWA [1], Samtools [2]
  • read extraction: bamtools [3]
  • de novo assembly: Velvet [4], BLAST [5]
  • Variant calling: Picard[6], GATK [7], effect annotation: snpEff [8]
  • Phylogenetic analysis: Clustalw. Phylip, EvolVew [9], [10], [11]
source("https://bioconductor.org/biocLite.R"); 
biocLite(c("Sushi", "data.table"))

libraries used

library(tidyverse)
library(data.table)
library(readr)
library(dplyr)
library(Gviz)
library(GenomicRanges)
library(Sushi)

Mapped reads versus total number of reads per sample

plot of chunk unnamed-chunk-2

NUMBER of SNP per sample

      POS REF ALT       GENE
1 2984740   A   G  RVBD_2666
2 3296843   A   G RVBD_2947c
3 1074558   G   A RVBD_0965c
4  414486   C   T  RVBD_0341
5 1636980   G   T RVBD_1452c
6 1636981   G   T RVBD_1452c

plot of chunk unnamed-chunk-3

Phylogeny of studies samples - samples 2, 9 and 15 show to be of different origin

plot of chunk unnamed-chunk-4

Result (Genbank submission and a paper)

  • Draft Genome Sequences of Two Drug-Resistant Mycobacterium tuberculosis Isolates from Myanmar.
  • Htin Lin Aung, Thanda Tun, Elizabeth Permina, Wint Wint Nyunt, Si Thu Aung, Kyi Kyi Thinn, John A. Crump, Gregory M. Cook.
  • Genome Announc. 2016 Sep-Oct; 4(5): e00850-16. Published online 2016 Oct 27. doi: 10.1128/genomeA.00850-16. PMCID: PMC5084853

supplementary