ExomeSeq Analysis

The ExomeSeq is the next level in medical exome sequencing offered by Eurofins Genomics India. The exome sequencing design provides >97% coverage of 22,000 genes, with a mean read depth of 100X. Most of the disease-associated genes are analyzed, having maximum coverage upto 100%  (depth coverage ≥20X) of all exons; which represents twice the number of genes with complete coverage offered by competitors, making it the most comprehensive exome sequencing test available. The ExomeSeq can be carried out for Proband, Trios and additional family member, which allows clinicians/researchers to choose relevant disease associated gene related to patient’s phenotype.

The human exome is the complete coding (exonic) region of the genome. It is estimated to encompass approximately 1-2% of the genome, yet contains approximately 85% of disease-causing pathogenic variants.

Current off-the-shelf exome kits used for genomic exome sequencing covers 92% of the exome. Traditionally, gene discovery has been done in research laboratories; however, now with the ability to sequence nearly the entire coding region of the human genome, it is possible for genomic laboratories/ Researchers to use this information to identify a previously unrecognized cause of disease.

Workflow and deliverables

a. Quality check of raw reads:

The raw reads will be subjected to quality filtration and adapter trimming. The primer sequences, poly(A) tails and reads produced from ribosomal DNA templates will be removed. The high quality data will be used for downstream analysis.

b. Alignment against Human Reference Genome.

The high quality reads are then mapped to, and analyzed in, comparison with the published human genome build UCSC hg19 reference sequence using bwa aligner. The targeted coding exons and splice junctions of the known protein-coding RefSeq genes are assessed for the depth of coverage and data quality threshold values.

c. Variant Calling

The Variant calling is performed using GATK variant pipeline, for the exome relevant to clinical indication.

d. Annotation

Clinically relevant mutations were annotated using published variants in literature and a set of variant databases including EmVClass (EGL's Variant Classification Tool), ClinVar, OMIM, GWAS, HGMD, dbSNP, COSMIC.

Deliverables

Exome for individual (Proband)

• Quality filtration of reads
• Alignment of reads to human reference hg19
• Summary statistics of coverage chromosome wise
• Complete list of mutation in excel (annotation of variants using various databases including 1000 genome, dbSNP, DBNSFP, HGMD, GWAS, COSMIC, ClinVar and EmVClass)
• Disease related mutation list in excel
• Comprehensive report with clinical importance.

Exome for family Trios

• Quality filtration of reads
• Alignment of reads to human reference hg19
• Summary statistics of coverage chromosome wise
• Complete list of mutation in excel (annotation of variants using various databases including 1000 genome, dbSNP, DBNSFP, HGMD, GWAS, COSMIC, ClinVar and EmVClass)
• Disease related mutation list in excel
• Pedigree analysis
• Comprehensive report with clinical importance.