Prerequisites

List of Available Utilities

The first most common step to analyze NGS datasets is to align the raw reads in fastq format to the reference genome using aligners of choice such as BWT,  Novoalign and Bowtie. The aligned reads in sequence alignment map (SAM) format can be converted to binary alignment map (BAM) format and sorted by genomic position by using tools such as samtools. PAINT then takes these sorted alignments in BAM format as input and determines the parental contributions in hybrids. Broadly, the steps involved are as follows: 

1) Determination of Somies. PAINT finds the average coverage values of all chromosomes in a sample. The average of average coverage values corresponds to the ploidy of the organism. Based on this rule, it determines the somy of each chromosome.

2) Extraction of Markers. PAINT finds SNVs using a custom built SNV caller. The SNV caller in PAINT determines the thresholds to call heterozygous SNVs based on the somy of the chromsome. PAINT first finds parental Homozygous SNVs against a common reference genome. It then filters out homozygous SNVs that are common to both the parental lines. The remaining SNVs represent loci where parental lines are different from each other.

3) Determination of Alleles and Allele Frequencies. PAINT finds The allele composition and allele frequency composition in the hybrids at the marker loci. Since the parental alleles are known and are homozygous different, their frequencies in the hybrids can be determined. Based on the somy of the chromosome, the parental contributions towards the hybrids can be determined accurately.

4) Summarizes NGS for easy depiction. PAINT summarizes the NGS data in a format that can be read by R scripts to depict in a format to study parental chromosomal contributions, aneuploidies and LOH events. See heatmap, bottle brush plots and line plots