Exome Sequencing

Exome sequencing allows more selective profiling of the genome, targeting only the coding regions, in contrast to whole-genome sequencing.  The CCCB uses the Agilent Sure Select XT2 technology to perform exome sequencing, using their whole exome bait pools. As many as 8 samples can be multiplexed pre-capture with whole exome bait pools, which are available for human and mouse. Sequencing may be performed with either Single Read (SR) or Paired End (PE) flowcells, depending on the type of analysis you wish to perform.

Sequencing Results

  • Raw Data:  Demultiplexed raw reads in compressed fastq format
  • Quality Control Report: Sequence data quality assessment based on FastQC package, including per-base read quality, over-represented sequences, and over-represented k-mers.
  • Alignment File: Sequenced reads are aligned to a reference genome to produce an aligned BAM file.  The BAM file may be subsequently filtered for duplicate reads (e.g. from PCR artifacts) and for unambiguously mapped, primary alignments.  Appropriate sorting and formatting may be added for integration with supplemental downstream analysis.

Standard Bioinformatics

  • Variant Calling: Variant calling is accomplished with the Genome Analysis Toolkit (GATK) HaplotypeCaller, which improves putative calls of small variants (SNPs, INDELS) via local de novo assembly.  Other standard tools such as samtools (http://samtools.sourceforge.net) or the GATK UnifiedGenotyper are possible.  Filtering of common variants can be performed with supplemental information provided by public databases (e.g. dbSNP).  Standard VCF (Variant Call Format) files are provided as output.
  • Variant Annotation: The SnpEff tool may be used to provide additional annotation and functional prediction of the variants to guide downstream inspection and analysis.  SnpEff is designed to work seamlessly with other bioinformatics packages such as the GATK.   

Customized Bioinformatics

We can also perform customized analysis to meet specific needs of your projects.

Required Sample Inputs

Agilent SureSelect XT2 on Fresh Frozen: 100 ng DNA
Agilent SureSelect XT2 on FFPE: 1 ug DNA