The updated content, enhanced capture uniformity, and an even further simplified workflow were designed to help accelerate your research. SeqCap EZ Human Exome Library v2.0 sets a new standard for exome sequencing by giving you the power to capture over 20,000 genes and sequence them on a single lane.

Minimize Sequencing Needs to a Single Lane

• Using actual capture and sequencing data, 2.1 million DNA probes have been redistributed to improve uniformity and reduce sequencing needs of exome capture to ~3 Gb of sequencing.

Comprehensive Coverage of RefSeq Coding Exons

• SeqCap EZ Human Exome Library v2.0 design utilizes the latest genomic content from RefSeq, CCDS and miRBase databases.
• Coverage of ~20,000 genes with single lane sequencing.

Easy-to-use, Scalable Protocol

• Workflow includes gel-free sequencing library preparation, single tube capture with stable DNA probes, and easy hybridization setup.

Figure 1

Figure 1: Empirical rebalancing improves uniformity and decreases sequencing needs. (A) Optimized Sequence Capture design utilized by Roche NimbleGen delivers better uniformity than a simple tiling approach. For SeqCap EZ Human Exome Library v2.0, an additional step called empirical rebalancing is used to redistribute probe density across exons based on sequence coverage, thus improving uniformity and decreasing the amount of sequencing needed. (B) Sequence coverage for the exons from FMR1 gene is shown for three solution-based exon capture methods: capture reagent from another vendor using simple tiling design, SeqCap EZ Exome v1.0 with optimized design, and SeqCap EZ Exome v2.0 with empirical rebalancing.

Table 1

Table 1: Exome capture performance with single lane sequencing. The exome from HapMap sample NA12762 was captured and sequenced with a single 2x75 paired-end lane. Data was analyzed using SOAP (Short Oligonucleotide Analysis Package). SNP calls from single lane data were compared to known HapMap SNPs for NA12762. Data shown are average and standard deviation from 16 experiments.

Figure 2

Figure 2. SNP discovery in exomes with single lane sequencing. The exomes from two HapMap research samples were captured and sequenced with a single paired-end lane. Data was analyzed using SOAP (Short Oligonucleotide Analysis Package) and SNP calls were compared with dbSNP131.

Roche NimbleGen offers SeqCap EZ Library as a solution-based method for target enrichment.

Sequence Capture Protocols

1. Genomic DNA: SeqCap EZ Oligo pool is made against target regions in the genome.
2. Library Preparation: Standard shot-gun sequencing library is made from genomic DNA.
3. Hybridization: The sequencing library is hybridized to the SeqCap EZ Oligo pool.
4. Bead Capture: Streptavidin beads are used to pull down the complex of capture oligos and genomic DNA fragments.
5. Washing: Unbound fragments are removed by washing.
6. Amplification: Enriched fragment pool is amplified by PCR.
7. Enrichment QC: The success of enrichment is measured by qPCR at control loci.
8. Sequencing-Ready DNA: The end product is a sequencing library enriched for target regions, ready for high throughput sequencing.

Download the complete 6-file set!

The design and annotation files provide information about genomic regions covered by the capture probes and the genes included in these regions. These files were designed for use with the following Roche NimbleGen products:

• SeqCap EZ Human Exome Library v2.0, 4 Reactions (Catalog No. 05860482001)
• SeqCap EZ Human Exome Library v2.0, 48 Reactions (Catalog No. 05860504001)

Overview

The SeqCap EZ Exome Library v2.0 product covers more than 20,000 genes in the human genome. The following sources provided information about the genes:

• NCBI Reference Sequence (RefSeq) RefGene from UCSC (January 2010)
• CCDS from NCBI (September 2009)
• miRNAs from miRBase (version 14, September 2009)
• Customer inputs

All the genome coordinates were based on human genome build GRCh37 (hg19).

For RefSeq genes, only transcripts with an “NM_” prefix were selected, and only protein coding parts of the transcripts were targeted. For exons that are smaller than 100 bp, Roche NimbleGen extended the target region to 100 bp.

The total size of the target regions is 36.5 Mb. Roche NimbleGen selected 2.1 million long oligo probes to cover the target regions. Because some flanking regions are also covered by probes, the total size of regions covered by probes is 44.1 Mb, larger than the initial target regions.

In the file descriptions provided in the next section, “target regions” refer to the 36.5 Mb targets selected from various databases, and “probe-covered regions” refer to the 44.1 Mb regions covered by long oligo capture probes.

File Descriptions

The Target_Regions folder contains two files:

• SeqCap_EZ_Exome_v2.gff * - There are two tracks in this .gff file. The primary_target_region track displays the 36.5 Mb target regions, and the capture_target track displays the 44.1 Mb probe-covered regions.
• SeqCap_EZ_Exome_v2.bed ** - There are two tracks in this .bed file. The target_region track displays the 36.5 Mb target regions, and the tiled_region track displays the 44.1 Mb probe-covered regions.

The Annotations folder contains four files:

• SeqCap_EZ_Exome_v2_annotations.xls - This Microsoft Excel file lists the genes and miRNAs that are targeted by the design. There are three worksheets in the file, listing RefSeq genes, miRNA genes, and other genes. The other genes include customer provided genes (using the UCSC Genes database) and CCDS genes that are not in the RefSeq database. Most column headers are self-explanatory. Two of the columns provide information about how well the specific exon target is covered by the capture probes:
  
  ARRAY COVERAGE = Percentage of target base covered by probes. Be aware that a region not covered by probes can still be captured if its neighboring regions are covered by probes. Refer to the following description of the ARRAY COVERAGE W 100BP EXTENSION column for more information.
  
  ARRAY COVERAGE W 100BP EXTENSION = Percentage of target base covered by probes or located within 100 bp to one or more probes. Because the DNA fragments captured by the SeqCap EZ Exome Library are generally greater than 200 bp, sequencing results typically show sufficient coverage of 100 - 200 bp flanking regions at both sides of a region targeted by probes. Therefore, coverage with 100 bp extension is a better estimate of how much of the target region will receive sequence coverage.
• SeqCap_EZ_Exome_v2_RefSeq.gff * - There is a single track in this .gff file. The track lists the original coordinates of the coding exon targets as determined from the RefSeq database (Jan 2010).
• SeqCap_EZ_Exome_v2_miRNA.gff * - There is a single track in this .gff file. The track lists the original coordinates of the miRNA targets as determined from miRBase database (v14).
• SeqCap_EZ_Exome_v2_other.gff * - There is a single track in this .gff file. The track lists customer provided genes (using UCSC Genes database) and CCDS genes (Sept 2009) that are not in the RefSeq database.

* The four GFF files above can be opened using SignalMap software. When using SignalMap software, each vertical bar represents one exon target, and you can move the cursor over each exon to display the accession number/sequence identifier.
** BED files can be displayed as a custom annotation track using the UCSC Genome Browser.

Download the complete 6-file set!

Use SeqCap EZ Hybridization and Wash Kits to perform hybridization and wash steps in sequence capture protocols using NimbleGen SeqCap EZ products. The kits are available in handy 24 and 96 reaction pack sizes.

Description	Catalog Number	Pack Size	Kit Capacity	Compatible Applications	Ordering*
SeqCap EZ Hybridization and Wash Kits	05634261001	1 Kit	24 Reactions	SeqCap EZ Library
	05634253001	1 Kit	96 Reactions
* Availability of products varies from country to country.

Search the SeqCap EZ Library - Literature for more information.