- NEXTFLEX® End-Repair & Adenylation Buffer Mix
- NEXTFLEX® End-Repair & Adenylation Enzyme Mix
- NEXTFLEX® Ligase Enzyme Mix
- NEXTFLEX® DNA Barcoded Adapter (25 μM)
- NEXTFLEX® PCR Master Mix
- NEXTFLEX® Primer Mix
- Nuclease-free Water
- NEXTFLEX® Resuspension Buffer
- NEXTFLEX® HYB1*
- NEXTFLEX® HYB2*
- NEXTFLEX® HYB3*
- NEXTFLEX® HYB4*
- NEXTFLEX® Barcode Blockers (250 μM)
- NEXTFLEX® Universal Oligo 1 (500 μM)
- NEXTFLEX® Block 1*
- NEXTFLEX® Block 2*
- NEXTFLEX® RNAse Block
- NEXTFLEX® XT Binding Buffer*
- NEXTFLEX® XT2 Hybridization Buffer**
- NEXTFLEX® Capture Binding Buffer**
- NEXTFLEX® Wash Buffer 1
- NEXTFLEX® Wash Buffer 2
- 100 mM NaOH**
- NEXTFLEX® Neutralization Buffer***Provided only in NEXTFLEX® Pre- & Post- Capture Combo Kit SureSelectXT®
**Provided only in NEXTFLEX® Pre- & Post- Capture Combo Kit SureSelectXT2®
REQUIRED MATERIALS NOT PROVIDED
- 100 ng – 1 μg of fragmented DNA in up to 32 μL nuclease-free water.
- Ethanol 100% (room temperature)
- Ethanol 80% (room temperature)
- Covaris System (S2, E210) and microTUBE Snap-Cap Kit (Cat # 520045)
- 1X Low TE Buffer (10 mM Tris-HCl (pH 8.0), .1 mM EDTA)
- Qubit fluorometer and Quant-iT dsDNA BR Assay Kit (Life Technologies, Cat # Q32850)
- 2100 Bioanalyzer and High Sensitivity DNA Kit (Agilent, Cat # 5067-4626)
- SureSelectXT Capture Library or SureSelectXT2 Capture Library (Agilent Technologies)
- 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
- 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar
- Nuclease free 1.5 mL snap-top centrifuge tubes
- Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
- Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63880)
- Dynabeads MyOne Streptavidin T1 (Life Technologies, Cat # 65601)
- Magnetic Stand -96 (Ambion, Cat # AM10027) or similar
- 2, 10, 20, 200 and 1000 μL pipettes / multichannel pipettes
- Nuclease-free barrier pipette tips
Pooling Recommendations for SureSelect Target Capture
Agilent’s SureSelect system is offered in iterations intended for single-plex capture as well as multiplex capture (SureSelect XT and SureSelect XT2, respectively). The SureSelect XT2 protocol provides specific recommendations on the number of samples to pool per capture, depending on the targeted region size. Roche Nimblegen’s SeqCap system also provides instructions for performing multiplex captures; however they do not provide the same rigid recommendations regarding the number of samples to pool as does Agilent. The number of samples to pool, or whether to pool at all, is best determined after taking many factors into account.
The ability to pool indexed samples prior to target capture has been shown to reduce hands-on processing time and reagent costs, while still allowing variant detection, albeit at the expense of reduced capture efficiency and depth of coverage [1, 2, 4]. Performing a single capture reaction on several pooled indexed samples reduces hands-on time in projects where simultaneous captures of pooled libraries can be completed in much less time than would be spent performing a capture on each individual library. For example, a project of 96 samples, where indexed libraries are pooled in groups of 12 prior to target capture, can be processed as 8 capture reactions prepared in parallel. The same project of 96 samples would be incredibly difficult to perform as 96 parallel target capture reactions without the aid of an automated liquid handler.
The reduction in capture efficiency seen when pooling samples prior to hybridization can be mediated by increasing the amount of data generated, e.g. longer read length, paired-end sequencing, etc. However, the cost of increased sequencing should be weighed against the alternative of performing individual captures, as well as the costs of potential sample failure. The potential need to re-process a single library and capture as opposed to re-processing a multiplex capture may reveal the more advantageous strategy.
Another disadvantage of pre-capture pooling of samples results from the re-amplification of the recovered library fragments after the capture reaction. PCR is known to recombine different template molecules at low levels, and in a traditional Illumina-style library multiplex PCR, this could cause ambiguity of sample origin upwards of 0.4% .
The frequency of incoming samples might also influence a user’s preference for pre-capture or post-capture pooling workflow. If all samples for a project are available at the same time, the time-saving benefit of pre-capture pooling can be fully appreciated. On the other hand, if samples will only become available one at a time over a period of time, the user may prefer to prepare sample libraries and perform individual captures as the samples become available.
Bioo Scientific offers improved complete solutions for library prep using target capture with both the Agilent SureSelect system and the Roche Nimblegen SeqCap system. Both methods possess the flexibility to perform pre-capture pooling or post-capture pooling. Please contact us at [email protected] to learn how to improve your target capture workflow with NEXTflex solutions.
1. Cummings et al. 2010. Combining target enrichment with barcode multiplexing for high throughput SNP discovery. BMC Genomics.
2. Kenny et al. 2010. Multiplex target enrichment using DNA indexing for ultra-high throughput SNP detection. DNA Research.
3. Kircher et al. 2011. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Research.
4. Shearer et al. 2012. Pre-capture multiplexing improves efficiency and cost-effectiveness of targeted genomic enrichment. BMC Genomics.
The NEXTFLEX® Pre- & Post- Capture Combo Kits contain enough material to prepare 16 or 96 DNA samples for Illumina® sequencing. The shelf life of all reagents is 12 months when stored properly. NEXTFLEX® Resuspension Buffer and Nuclease-free Water should be stored at room temperature. All other components can be safely stored at -20°C. This kit is shipped on dry ice.