Enrichment - Complete sample preparation to sequencing workflow

Deep sequencing of specific regions in a large number of samples

Your enrichment project

Systematic identification of rare and common SNPs, insertions, and deletions in specific regions to study genetic variation between individuals and their correlation with specific phenotypes and diseases.

Strategy

The possibility to enrich target regions ranging from smaller than 200 Kb to more than 50 Mb in virtually any genome of interest enables a highly flexible project design - from small projects to up to thousands of samples - and eliminates the setting up of thousands of PCRs. Create your own custom designs for focused analyses of individual, specific candidate loci. Use pre-defined bait designs to perform comprehensive kinome and exom studies in human or the classic model vertebrate mouse. Optionally expand and individualize existing catalog enrichment kits through adding of additional customized content. Enrichment is performed with the robust and optimized in-solution sequence capture method using long high-quality RNA baits for the hybridization.

Starting material

Genomic DNA

Library preparation

Library preparation from genomic DNA for Illumina HiSeq 2000

Enrichment

Optional: Design of the baits using the Agilent eArray online design tool
Enrichment using the most suitable Agilent Technologies system:
- SureSelect Target Enrichment System (from 200 Kb to more than 5 Mb)
- SureSelect Human All Exon (predesigned plus optionally customized)
- New: SureSelect Human Kinome
- New: SureSelect Mouse All Exon
Hybridisation
Capture of enriched region
Multiplexing (optional) to mix various samples for subsequent sequencing

Sequencing

Illumina HiSeq 2000 I single or paired end reads for detection of SNPs and small InDels
At least 30x coverage depending on the project and sample source

Bioinformatics

Preprocessing (bam and bedgraph files)
- Quality check of sequence data
- Alignment to reference
- Read grouping
- PCR duplicate removal
- Localized realignment
- Base quality score recalibration

Variant detection
- Calling single nucleotide polymorphisms (SNPs): known, novel and ranked SNPs (tsv file), all SNPs (vcf file)
- Calling deletion / insertion polymorphisms (DIPs; up to 5 bp); InDels (tsv and vcf file)
Post processing
- Annotation
- Variant filtering
- Variant ranking
- Coverage computing
- Data summary (png, pdf, txt)