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The identification of common and rare variants in the human genome is essential to better understanding the etiology of complex human diseases. Targeted resequencing allows researchers to focus efficiently and cost-effectively on regions of interest, for instance the protein coding portion of the genome, the exome, or custom candidate regions from 100 kb to up to 60 Mb.

The TargetSeq™ Exome and Custom Enrichment System provides efficient targeted sequencing.

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Figure 1. In-solution hybridization method for exome capture enables multiplexing prior to capture.
  • Targeted—focused, high-density probe design
  • Economical—precapture sample multiplexing
  • Optimized—superior 5500 Series Genetic Analyzers and SOLiD® Systems
  • Integrated—streamlined data analysis with LifeScope™ Genomic Analysis Software
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Figure 2. Integrated, targeted resequencing workflow featuring TargetSeq™  Enrichment Kits and the 5500 Series Genetic Analysis Systems.

 

Exome sequencing is best performed using the TargetSeq™ Exome and Custom Enrichment System kits, which are based on an in-solution hybridization method for exome capture (Figure 1). The kits are optimally designed to seamlessly integrate into the 5500 Series Genetic Analysis Systems workflow (Figure 2).

Over 2 million probes of exome content developed at Baylor College of Medicine

The TargetSeq™ Exome and Custom Enrichment Kits are designed to an expanded exome target of 37.3 Mb that is focused on relevant content, including both predicted and validated genes permitting capture of >98% VEGA [1] and CCDS [2] coding regions, and RefSeq exons [3]. The capture region of 45.1 Mb is covered by over 2 million capture probes. Other exome enrichment solutions are designed to capture targets of 50 or 62 Mb with fewer than 400,000 probes! The smaller capture region of the TargetSeq™ Exome Enrichment design focuses sequence throughput on exons, rather than additional content, to target your budget to what matters most.

The TargetSeq™ Exome Enrichment design contains a much larger number of probes that overlap, which have been distributed empirically to optimize capture efficiency and uniformity across the exome. The high density of probes (on average ~8 probes tile each targeted region) in the TargetSeq™ Exome Enrichment design provides more opportunities for productive hybridization reactions to occur and facilitates even target coverage.

Thousands of human exomes have been sequenced at the Baylor College of Medicine’s Human Genome Sequencing Center as they developed and optimized the design of the TargetSeq™ Exome Enrichment Kits.

Pre-capture multiplexing provides economical enrichment

Sample multiplexing is achieved by pooling just 125 ng of each of 4 barcoded fragment libraries prior to target capture. The results are robust, even coverage of each of the barcoded libraries, with cost-savings realized by reducing the number of captures needed. Additionally this pre-capture multiplexing facilitates higher sample throughput by simplifying sample preparation for sequencing.

Optimized for superior performance

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Figure 3. Click to enlarge.

The TargetSeq™ Exome and Custom Enrichment design was developed specifically for optimal performance with SOLiD® chemistry, resulting in superior uniformity of coverage across targeted region. The uniformity of on-target bases of several exome enrichment solutions is compared in Figure 3, where the TargetSeq™ Exome Enrichment sample (in blue) exhibits a sharper peak, representing a higher number of on-target bases at a depth of coverage near the mean (high uniformity). In comparison, enrichment solutions A and B lack a defined peak and have long tails, representing a large variance in depth of coverage (low uniformity). High uniformity in coverage minimizes the amount of sequencing you need to do to get a desired threshold of coverage (e.g., 20X coverage) over a good fraction of your target bases (e.g., 75–90%).

Figure 3 (above right). The results from a single sample enriched with the TargetSeq™ Exome and Custom Enrichment Kit, run in a single lane on the 5500xl Genetic Analyzer and analyzed with LifeScope™ 2.0 Genomic Analysis Software is shown in Table 1. The 85.4% target bases covered at greater than or equal to 20x coverage and just 3.36% target bases not covered demonstrate the excellent coverage of the target region. A total of 21,916 exonic SNPs were detected in the target region and of these 19,939 (91%) occur in dbSNP. We have consistently observed >90% SNP concordance rates with dbSNP and have found higher numbers of exonic SNPs detected than with other enrichment solutions (data not shown).

Integrated workflow with LifeScope™ Genomic Analysis Software for data analysis

The workflow for exome sequencing is integrated from library construction & exome enrichment, through to data analysis with automation options, to increase throughput and reduce hands-on time. Analysis is simplified with LifeScope™ Genomic Analysis Software the targeted sequencing pipeline that streamlines your results with reports, annotations and visualizations.

Table 1. Excellent exome sequencing results.

Sequencing Statistics

Raw reads Reads mapped Percent mapped reads in targets Fold enrichment
128,180,855112,340,30472.23%39.4

Coverage

Target bases not covered Target bases covered at
1x or more
Target bases covered at 10x or more Target bases covered at 20x or more Average depth of coverage
3.36%96.64%91.65%85.40%92.0

Variants

Type of variant Number of variants Percent variants in dbSNP
SNPs31,59295.73%
Homozygous SNPs11,73899.58%
Heterozygous SNPs19,85493.46%
InDels2,58586.73%
Homozygous InDels80996.04%
Heterozygous InDels1,77182.46%

The highly characterized NA 12878 HapMap sample was used to create a fragment library, enriched with TargetSeq™ Exome and Custom Enrichment Kit, sequenced with 75 bp reads on a single lane of a 5500xl Genetic Analyzer and analyzed with LifeScope™ Genomic Analysis Software. The output below is provided by analysis with LifeScope™ 2.0 Genomic Analysis Software where the primary target BED file has been used as the reference with automated 50 bp padding as well as hg19, and dbSNP132.

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