1. First Bite of Variant Calling in NGS/MPS Precourse materials
Yonglan Zheng

2. Precourse materials General NGS variant calling workflow [slide#3]
GATK Best Practice as an example
NGS file format [slides#4-8]
NGS variant calling tools and platform [slided#9]
FastQC, BWA-MEM, Picard (Markduplicates), GATK (RealignerTargetCreator, IndelRealigner, Unified Genotyper), SnpEff, Freebayes

3. GATK Best Practice (v3.x)
Multi-sample calling is replaced by a winning combination of single-sample calling in gVCF mode [Genome VCF (gVCF) for both variant and non-variant positions] and joint genotyping analysis.

4. FASTQ
A FASTQ file (.fq and .fastq) is a text-based file for storing both a biological sequence (usually nucleotide sequence) and its corresponding quality scores.
Each entry in a FASTQ file consists of four lines:
• Sequence identifier
• Sequence
• Quality score identifier line (consisting of a +)
• Quality score
Quality
A quality value Q is an integer mapping of p (i.e., the probability that the corresponding base call is incorrect). Phred quality score:
Sequence identifier
@<instrument>:<run number>:<flowcell ID>:<lane>:<tile>:<x-pos>:<y-pos> <read>:<is filtered>:<control number>:<index sequence>

5. FASTQ An example of a valid entry is as follows:
@EAS139:136:FC706VJ:2:5:1000: :Y:18:ATCACG
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA +

6. SAM/BAM/CRAM
A SAM (Sequence Alignment/Map) file (.sam) is a tab-delimited text file that contains sequence alignment data. A BAM file (.bam) is the binary version of a SAM file. Typically CRAM achieves 40-50% space saving over the alternative BAM format. It uses reference based compression, meaning that only base calls that differ to a designated reference sequence need to be stored.
Headers
Alignment section: mandatory fields
CIGAR (Compact Idiosyncratic Gapped Alignment Report) String
Alignment section: optional fields

7. VCF/BCF
A VCF (Variant Call Format) file (.vcf) is a text file that contains meta-information lines (prefixed with ”##”), a header line (prefixed with ”#”), and data lines each containing information about a position in the genome and genotype information on samples for each position (text fields separated by tabs). VCF’s binary counterpart is BCF.

8. MAF
A Mutation Annotation Format (MAF) file (.maf) is a tab-delimited text file that lists mutations. The format originates from The Cancer Genome Atlas (TCGA) project. Its columns include:
Hugo_Symbol, Entrez_Gene_Id , Center, NCBI_Build, Chromosome, Start_Position, End_Position, Strand, Variant_Classification, Variant_Type, Reference_Allele, Tumor_Seq_Allele1, Tumor_Seq_Allele2, ...
BED
A BED (Browser Extensible Data) (.bed) file is a tab-delimited text file that defines a feature track. It consists of one line per feature, each containing 3-12 columns of data.
Required
Optional

9. NGS Variant Calling Tools and Platform
FastQC:
BWA-MEM:
Picard:
Picard MarkDuplicates:
GATK:
GATK RealignerTargetCreator:
GATK IndelRealigner:
SnpEff:
Freebayes:
Galaxy: (main site); (test site)