SeqCap EZ Choice Library
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Format: 2.1 million long oligonucleotides in solution Source: UCSC Build: HG19 or HG18 Design: Based on researcher provided target regions. Automation: An automated solution for SeqCap EZ Choice Library using the Caliper Sciclone NGS workstation is now available. |
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| Description | Target Region | Volume per Reaction | Catalog Number | Pack Size | Work- flow |
Ordering* |
| SeqCap EZ Choice Library | 100kb-7Mb human genomic regions. | 4.5 µl | 06266282001 | 12 reactions |  |
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| 06266304001 | 24 reactions | |
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| 06266312001 | 48 reactions | |
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| 06266339001 | 96 reactions | |
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| 06266347001 | 384 reactions | |
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| 06266355001 | 960 reactions | |
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| * Availability of products varies from country to country. | ||||||
Advantages
- Achieve uniform capture, and save on sequencing costs. The NimbleGen Sequence Capture design algorithm utilizes up to 2.1 million probes and ensures high-performance capture.
- Capture up to 50Mb custom regions with a single design.
- Identify more mutations. More complete coverage of target regions with advanced repeat masking methods.
- Easy workflow with stable DNA probes.
Figure 1
Figure 1. NimbleGen Sequence Capture design utilizes many more probes than other methods, and this brings important advantages for capture performance. Simple Tiling Designs (Panel A) offer up to 55,000 120mer probes (baits) tiled across the region. NimbleGen Sequence Capture Designs (Panel B) offer up to 2,100,000 50-105mer probes selected for the region using a special algorithm. This dramatic increase in probes (38X) offers the following benefits:
• More attempts for every target base
• Redundancy to reduce risk
• Ability to redisribute probes for better uniformity
Figure 2
Figure 2. Comparing to simple tiling design, probes are relocated in the Sequence Capture design to improve capture uniformity.
Applications
Figure 3
| Sample Name | Total Reads | On Target (%) | Het True Positive Rate | Average Coverage | Raw Coverage | Bases Required for 20X Coverage |
| 500k-3 | 91,004 | 58.58 | 0.950 | 16.54 | 37.08 | 20,182,861 |
| 250kb1-2 | 76,758 | 57.64 | 0.916 | 13.56 | 51.08 | 11,837,439 |
| 250kb1-3 | 86,209 | 58.49 | 0.926 | 16.21 | 57.59 | 11,665,413 |
| 250kb2-5 | 92,577 | 46.23 | 0.958 | 43.52 | 61.14 | 10,815,531 |
| 100kb1-1 | 55,978 | 53.23 | 1.000 | 27.66 | 92.02 | 4,219,538 |
| 100kb2-1 | 44,617 | 66.43 | 0.965 | 25.88 | 96.09 | 3,493,572 |
| 100kb2-3 | 90,726 | 65.88 | 1.000 | 68.67 | 204.36 | 3,522,738 |
| 100kb3-2 | 90,586 | 64.79 | 0.897 | 58.23 | 182.87 | 3,458,960 |
Figure 3. Targeted sequencing results with SeqCap EZ Choice in combination with 454 Rapid Library protocol. Top panel, specificity of capture for 8 different designs (the data are the average of 3 captures for each design, ±1 standard deviation). Bottom panel, heterozygous SNP detection (gray column) for known HapMap SNPs.
Figure 4
Figure 4. Targeted sequencing results with SeqCap EZ Choice in combination with Illumina paired-end library protocol. Specificity of capture for 10 different designs are shown (the data are the average of 3 captures for each design, ±1 standard deviation).
Figure 5
| Design (both have ~250kb target sizes) | Exonic Regions | Contiguous Region |
| Target bases covered by probes | 95.6% | 78.8% |
| Target bases covered by at least 2 reads | 95.3% | 82.4% |
| Target bases covered by at least 8 reads | 93.4% | 75.4% |
| Sensitivity of calling known HapMap SNPs | 98.3% | 91.7% |
| Specificity of calling known HapMap SNPs | 98.9% | 100% |
Figure 5. Targeted sequencing results for exons and contigs. HapMap samples were captured and sequenced with GAIIx.
Protocol
Roche NimbleGen offers two types of capture methods: SeqCap EZ Library, a solution-based method and Sequence Capture Arrays, an array-based capture method.
Sequence Capture Protocols
- Genomic DNA: SeqCap EZ Oligo pool or an array is made against target regions in the genome.
- Library Preparation: Standard shot-gun sequencing library is made from genomic DNA.
- Hybridization: The sequencing library is hybridized to the SeqCap EZ Oligo pool or to the Sequence Capture array.
Steps 4 and 5 are different for each protocol:
SeqCap EZ Library, biotinylated DNA oligos in solution
- Bead Capture: Streptavidin beads are used to pull down the complex of capture oligos and genomic DNA fragments.
- Washing: Unbound fragments are removed by washing.
Sequence Capture, capture probes synthesized on array:
- Washing: Unbound fragments are removed by washing.
- Target Fragment Elution: The enriched fragment pool is eluted and recovered from the array.
- Amplification: Enriched fragment pool is amplified by PCR.
- Enrichment QC: The success of enrichment is measured by qPCR at control loci.
- Sequencing-Ready DNA: The end product is a sequencing library enriched for target regions, ready for high throughput sequencing.
Reagents
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. | |||||
