Swift Hybridization Capture KitsMary Lukens2019-07-16T16:59:04+00:00
Swift Hybridization Capture Kits
Our Kits Save Sequencing Costs by Targeting Only Genes of Interest
Swift Hybridization Capture Kits, enable enrichment and sequencing of the human exome or subsets of disease-related genes involved in cancer and inherited diseases. Our kits save sequencing costs by targeting only genes of interest using a 4-hour capture, while delivering the breadth and depth of coverage required for comprehensive yet sensitive genomic profiling.
The high quality of Swift panels, combined with optimized hybridization reagents and protocols, results in a substantial improvement in on-target mapping rate and coverage uniformity compared to other commercially available panels. The panels are designed to allow variants to be called using fewer reads, and therefore, at lower costs.
These products include gene-specific capture probes and supporting hybridization and wash reagents, including beads and blockers. When used in conjunction with Swift’s portfolio of library preparation kits including library adapters with molecular identifiers, labs have a variety of complete workflow options to convert input DNA into targeted Illumina®-compatible libraries in a 1.5-day workflow.
Enables enrichment of the human exome or subsets of disease-related genes
Superior on-target performance and comprehensive coverage of human coding sequences from the RefSeq database, probes designed to version hg19
Saves sequencing costs
Pre-capture multiplexing facilitates orders of magnitude more efficient next generation sequencing by targeting genes of interest while conserving enrichment reagents
Provides high quality data
Probes achieve deep and uniform coverage even across GC-rich regions such as first exons
The Exome Panel consists of 429,826 individually synthesized, and quality controlled Swift Probes. The Exome Research Panel spans a 39 Mb target region (19,396 genes) of the human genome and covers 51 Mb of end-to-end tiled probe space. All probes in the panel are manufactured using GMP standards. Mass spectrometry and OD measurements are taken for each probe to ensure appropriate representation of the correctly manufactured probes in the pool.
The Pan-Cancer Panel consists of 7816 Swift Probes, spanning 800 kb of the human genome, for the enrichment of 127 significantly mutated genes implicated across 12 tumor tissue types for deeper sequencing coverage.
The Inherited Diseases Panel enables deeper sequencing of genomic regions containing genes and SNPs associated with inherited diseases. The gene list is based on the HGMD® (Human Gene Mutation Database) repository of known inherited disease-causing mutations.
Sample Types and Applications
Detection of germline inherited SNVs and Indels
Low frequency somatic variant detection of SNVs and Indels
What are the fragment size ranges for working with this protocol?
For optimal results, we recommend using fragmented DNA between 150-350 bp.
What is the shelf life of the Swift Hybridization Capture Kits?
The shelf life of the Swift Hybridization Capture Kits is at least 6 months from the time the product is delivered when stored at -20C and handled according to the protocol.
Can I use my favorite polymerase to amplify libraries prior to hybrid capture?
No, This protocol has been validated with KAPA HiFi HotStart ReadyMix from Kapa Biosystems (Cat# KK2601). We recommend performing the pre-capture PCR using KAPA polymerase to produce at least 500 ng of each prepared library for hybridization capture.
What is the recommended method for quantifying amplified libraries prior to performing hybridization capture?
Pre-capture libraries can be quantified by Qubit and assessed for library size on the Bioanalyzer.
How many libraries can be multiplexed into each hybridization capture?
For hybridization capture, multiplexing has been tested on up to 12 libraries (6 µg total DNA) with limited impact on data quality.
How do I select the appropriate blocking oligos for my libraries?
Using the correct blocking oligos can significantly improve overall capture performance. Swift blocking oligos are compatible with libraries and can be readily used with other Swift hybridization capture products. These blockers are designed for both single and dual indexing strategies when the index sequences are 6 to 8 bases in length or when using the Swift 9 base molecular identifiers (MIDs, Cat. No. 27024, 27096). Contact Swift SupportSM, for compatibility with alternate adapter sequences or indexing strategies.
What is the minimum amount of library required for capture?
We recommend using 500 ng of each prepared library for hybridization capture. For exome captures, Using less input for capture can result in higher duplicate rates, lower mean coverage, and poor coverage uniformity.
What is the most optimal method to concentrate libraries with blocking oligos prior to hybridization capture?
For optimal results, use a SpeedVacTM system (Savant) for concentrating DNA. Although the optional Appendix: AMPure XP Bead DNA concentration protocol can be used, our testing has found reproducible, though minor, adverse impact on GC bias during bead-based concentration.
Note: To multiplex a high quantity of samples, we recommend using a SpeedVac system; if you require a quicker turnaround, you may prepare the DNA samples following the instructions in the Appendix for the bead-based DNA concentration protocol.
What Cot DNA should I use if my DNA samples are non-human?
If your experiment focuses on non-human library captures, realize that Human Cot DNA included in the Swift Hybridization and Wash Kits might not be optimal. For the best results, use alternatives like mouse Cot DNA, or Salmon sperm DNA.
When and how do you recommend using the plate protocol?
We recommend using the plate protocol when processing more than 6 captures. Using the plate protocol (versus the tube protocol) shows lower sample-to-sample variability within one experiment. When working with plates, avoid using the wells on the perimeter of the plate’s edges. The plate protocol is optimized for a maximum of 4 columns of captures in standard 96-well plate format. We do not recommend running more than 32 captures at a time because the timing and temperature of washes will be impacted.
What is the optimal hybridization reaction incubation time?
We recommend a 4 hour hybridization incubation duration which the hybridization reaction tube or plate should be kept tightly sealed. Extending hybridization incubation time from 4 to 16 hours may improve panel performance, particularly for GC-rich or smaller panels (<1000 probes).
What types of standard blocking oligos are needed and what are their function?
They are two major issues when capturing DNA with the Swift probes.
The contribution of the repeat elements (I) even if you do not design your probes to target these sequences. The repeat can be within the fragment that you capture pulling down off-target material.
The other problem is with the adapter-adapter hybridization (II). The adapters are single sequence oligos that have the ability to hybridize to other adapters pulling down off-target DNA regions.
The solutions are:
I) Cot-1 DNA hybridizes to the repeat elements and both to your target as well as the off target regions inhibiting the pulling down of the off target fragment with the on-target fragment
II) Swift blocking oligos are single oligo sequences. They are complementary to the TruSeq adapter sequences and have a 3’ modification to inhibit extension. They bind to adapter sequences to inhibit the hybridization of the adapters to one another.
Do I need extra Cot DNA if performing the optional AMPure XP Bead DNA concentration protocol?
Yes, understand that the optional Appendix: AMPure XP Bead DNA concentration protocol requires more Human Cot DNA than the standard SpeedVac method outlined in the protocol. Additional Human Cot DNA can be purchased through IDT (IDT Cat# 1080768; 1080769). Using the standard amount of Human Cot DNA with AMPure XP Bead DNA concentration protocol can lead to lower flanked on-target percentage due to loss of small human Cot DNA fragments (50-300 bp fragment size) during AMPure XP concentration.
What are the critical steps to avoid capture failure?
Preheat the “heated wash buffers” a minimum of 15 minutes before use. Preheating helps ensure there is sufficient time to heat the buffers to 65 ºC at critical steps during the protocol.
Vortex every 10 to 12 minutes during the 45-minute bead capture to improve the kinetics of the capture.
Do not let the Streptavidin beads dry out at any point during the protocol. If necessary, extend washes slightly rather than let the beads dry out.
Ensure the Streptavidin beads remain fully re-suspended during room temperature washes. Vortex vigorously and adhere to the incubation guidelines during room temperature washes to improve data quality.
Always use fresh seals in each step of the protocol that calls for adhesive seals on plates. Using fresh seals avoids possible sample cross-contamination during the plate protocol.