- NEXTflex Rapid DNA-Seq protocol reduces library prep time
- Index-specific blocking oligos
- Higher percentage of on-target reads
- Options available for multiplex and single-plex captures
- Flexible multiplexing options with up to 96 barcodes for multiplexing
The NEXTflex™ Pre- and Post- Capture Combo Kit (Agilent SureSelectXT OR SureSelectXT2 Compatible) is a complete Illumina -compatible library prep solution designed for DNA library preparation, multiplexing, and barcode blocking to be used with Agilent SureSelectXT or SureSelect XT2 target enrichment systems. This kit can be used with any of Agilent’s SureSelectXT or SureSelectXT2 custom and pre-defined panels including the exome, kinome and inherited disease panels.
The NEXTflex Pre- and Post- Capture Combo Kit (Agilent SureSelectXT or SureSelectXT2 Compatible) is designed to prepare single, paired-end, and multiplexed genomic DNA libraries for DNA and exome target capture and Illumina compatible sequencing. This kit contains everything needed for library prep, hybridization, and post capture amplification with Agilent’s SureSelectXT or SureSelectXT2 target capture baits.
Integration of the NEXTflex Pre- & Post- Capture Combo Kits with SureSelectXT or SureSelectXT2 Target Capture Baits
- NEXTflex Rapid DNA-Seq library prep reagents
- Indexed barcodes
- Hybridization and wash buffers
- Post-capture amplification reagents
Fast Library Prep
The NEXTflex Pre- and Post- Capture Combo Kit (Agilent SureSelectXT or SureSelectXT2 Compatible) incorporates the NEXTflex™ Rapid DNA-Seq Kit, a rapid Illumina® DNA library prep kit producing libraries from as little as 100 ng of DNA in only two hours.
Higher % of On-target Reads
The NEXTflex Pre- and Post- Capture Combo Kit (Agilent SureSelectXT or SureSelectXT2 Compatible) for Illumina sequencing contain index-specific blockers, which produce a higher percentage of on-target reads than generic blockers.
Options available for Multiplex Captures and Single-plex Captures
The NEXTflex Pre- and Post- Capture Combo Kit (Agilent SureSelectXT Compatible) is recommended for users who plan on performing separate captures on each sample individually. For users who prefer pooling several libraries for fewer capture reactions, we recommend the NEXTflex Pre- and Post- Capture Combo Kit (Agilent SureSelectXT2 Compatible).
Flexible Multiplexing Options
NEXTflex™ DNA Barcodes and NEXTflex™ DNA Barcode Blockers are included in the NEXTflex Pre- and Post- Capture Combo Kit. The blockers are designed to fully block each indexed adapter strand, yielding higher %-on target reads. The NEXTflex Pre- and Post- Capture Combo Kits offer enhanced flexibility by allowing researchers to multiplex 16, 32, 48, 62, 78 or 96 samples depending upon their specific needs.
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.
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
NEXTflex™ Resuspension Buffer
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
Improved Library Prep Offers Higher Percentage of On-target Reads and Better Coverage for SureSelectXT2 Target Capture
Next-Generation Sequencing (NGS) has provided the scientific community with a powerful tool to sequence large and complex genomes in far less time than traditional Sanger sequencing methods. Even so, we have not yet reached a point in which sequencing and analyzing the 3.2 billion base human genome is either time or cost effective. Target capture strategies have been shown to provide a solution to this problem by reducing genome complexity to what is relevant to the question being asked. Target capture approaches require whole genome library preparation followed by systematic target selection through probe hybridization to genomic regions of interest followed by massively parallel sequencing. The size and complexity of the capture is user dependent but can range from large scale targeting of all protein coding regions (~50 Mb) to a subset of genes known to be involved in specific biological functions (<10 Mb). Pre-capture pooling of multiple samples prior to target capture is a cost effective method to maximize the amount of data obtained for multiple samples in a single sequencing run. Several target capture technologies are commercially available, all of which have their individual strengths and weaknesses. Agilent’s SureSelectXT2 system offers a wide range of probe sets including All Exon options for many different model organisms, as well as a flexible custom option allowing users to design their own capture panels, all of which can be purchased independent of upstream and downstream reagents required to construct sequence-ready capture libraries.
Target capture performance is dependent on several variables; however, one important factor is the barcode blocking strategy employed. Barcode blocking oligos bind to platform binding adapter sequences to reduce non-specific probe hybridization. A problematic class of unwanted molecules arise during in-solution hybridization target capture from the annealing of unrelated DNA sequences at complimentary terminal adapter sequences during probe hybridization. Identical adapter terminal sequences flank each template molecule in the hybridization pool. Thus, this molecule and its complement are in very high concentration relative to other molecules in solution. The joining of non-complementary template molecules at either terminal adapter sequence can be as little as two molecules, or a chain of many different unrelated sequences. Consequently, the presence of a bait binding site on one of the linked molecules will pull down the entire linked chain, resulting in a larger number of contaminating molecules and increasing the number of off-target reads in target capture sequencing results.
Bioo Scientific now offers all reagents required upstream and downstream of probe hybridization (Table 1) to prepare sequence-ready, multiplexed capture libraries compatible with all SureSelectXT2 bait sets. NEXTflex barcode blocker technology allows for maximum binding efficiency through index-specific blocking oligos, greatly reducing the number of off-target reads encountered in small and large target capture assays. Furthermore, utilization of NEXTflex reagents with SureSelectXT2 bait set improves target capture efficiency through robust library preparation workflow as well as important hybridization efficiency improvements.
Table 1. Integration of the NEXTflex Pre- and Post- Capture Combo Kit with SureSelectXT2 Target Capture Baits
Library Preparation and Target Capture
Genomic DNA was isolated from human blood cells of a consented individual and sheared to an average size of 200 bp. Library prep was performed using 100 ng of the same input DNA for each capture using either Agilent’s SureSelectXT2 Reagent Kit or Bioo Scientific’s NEXTflex™ Pre- and Post-Capture Kit, and barcoded adapters from each, where stated. Target capture was performed according to the manufacturer’s instructions using either all Agilent or all NEXTflex reagents, where stated. All baits sets used (SureSelectXT2 Human All Exon V5 and SureSelectXT2 Inherited Disease) were supplied from Agilent. Quality and quantity of capture libraries was determined using the Agilent 2100 Bioanalyzer. Normalized libraries were clustered on-board, and 150 bp paired-end sequencing was performed on the HiSeq 2500 across 4 lanes of 2 flow cells using rapid run mode.
NEXTflex Barcode Blocking Efficiency
Various barcode blocking strategies can be employed to reduce the presence of unwanted templates following target enrichment. Blocking strategies commonly used during in-solution hybridization assays bind the entirety of a designated strand, usually the common adapter, barcoded adapter, or both. Cost-effective configurations to block specific barcoded adapter sequences are randomized or deoxyinosine bases that barcode specific sequences. Additionally, manufactures may pool all blockers corresponding to the series of index sequences that they offer commercially, which would result in a substantially higher chance of index-specific blocking during hybridization than randomized or deoxyinosine bases. NEXTflex barcode blocker technology uses “index-specific” barcode blocking to most effectively block both adapter termini from annealing during hybridization while Agilent uses the “multiplexed blocker pool” strategy.
In this experiment (Figure 1) the efficiency of two new barcode blocking configurations were tested. The efficiency of an “index-specific” blocking strategy was tested by pre-capture pooling eight individually barcoded libraries and blocking with an oligo specific for the non-barcoded adapter (universal) and the respective index specific blockers. Pre-pooled libraries barcoded with indices 1-8 were blocked with a pool consisting of barcode blockers 1-8 and the universal blocker. This “index-specific” blocking configuration was compared to a “multiplexed blocker pool” strategy in which eight individually barcoded libraries were pre-capture pooled and blocked with a pool consisting of the universal blocker and 96 different blockers, eight of which were the exact complement to the eight barcoded libraries pooled prior to hybridization to Agilent’s SureSelectXT2 Human All Exome Target Enrichment V5 bait set (Figure 1).
Figure 1. Experimental design flowchart for NEXTflex libraries using different barcode blocking strategies prior to SureSelectXT2 all exon V5 target capture.
Of the 50.18 Mb of sequence targeted in the exome capture, both 8-sample captures had roughly the same amount of raw reads with ~170 million reads mapping to the hg19 genome and ~4% of all mapped reads calculated to be duplicates (Table 2).
Table 2. Mapping and coverage statistics for NEXTflex libraries using different barcode blocking strategies prior to SureSelectXT2 all exon V5 target capture. The NEXTflex Pre- & Post- Capture Combo Kit incorporates the index specific blocking strategy.
Of the two blocking strategies, the index-specific blocking performed better than the multiplexed blocker pool with reads mapping to target percentages of 69.3% and 65.5%, respectively. Index-specific exome capture produced a mean coverage of 14X while the multiplexed blocker pool approach produced 13X mean coverage of the 50.18 Mb targeted exome sequence. Index-specific blocking showed less variation in blocking efficiency of all 8 capture libraries than the multiplexed blocker pool, resulting in more uniform coverage of the target genome than alternative blocking configurations. The high percentage of total reads mapping to the target sequence demonstrates the effectiveness of index-specific blocking, yielding a higher proportion of on-target reads and increased coverage of genomic regions of interest with less sequencing.
Comparison of Agilent SureSelectXT2 Reagent Kit to NEXTflex Pre- & Post- Capture Kit
A head to head comparison between all Agilent and all NEXTflex reagents was also performed upstream and downstream of probe hybridization with the SureSelectXT2 bait set (Figure 2).
Figure 2. Experimental design flowchart for both all Agilent and all NEXTflex pre and post-capture reagents using the SureSelectXT2 Inherited disease bait set.
Based on the improved blocking performance demonstrated in the exome capture, index specific blocking was used for the NEXTflex capture and the supplied SureSelectXT2 blocking mix was used for the Agilent captures. Both Agilent and NEXTflex captures produced roughly the same amount of raw reads and both shared ~56 million reads mapping to the hg19 reference genome and 95% of the mapped reads were unique (Table 3).
Table 3. Mapping and coverage statistics for both all Agilent and all NEXTflex pre- and post-capture reagents using the SureSelectXT2 Inherited disease bait set.
Importantly, the capture library prepared using all NEXTflex reagents showed better performance, with 83% of reads mapping to the targeted sequence compared to 78% on-target for the Agilent control. Furthermore, the NEXTflex capture pool produced a mean coverage of the target sequence at 151X compared to 137X attained by the Agilent control capture.
The seamless compatibility between the NEXTflex Pre- and Post- Capture library preparation kit and all reagents required for target capture using Agilent’s SureSelectXT2 bait set was demonstrated. The advantages of using NEXTflex reagents for SureSelectXT2 target capture experiments begin with the minimal hands on time and enhanced ligation technology offered by our library prep kit, which delivers more unique reads and higher mean coverage per sample compared to competitors. Importantly, the index specific barcode blocking strategy offered by Bioo Scientific outperforms alternate blocking configurations, resulting in better overall target capture performance with an increased number of bases on target. Agilent’s SureSelectXT2 probe sets can be purchased individually and all other components required for target capture experiments can be purchased from Bioo Scientific in convenient 16 or 96 reaction packages offering greater multiplexing flexibility. Currently, Bioo Scientific offers 96 8-nucleotide barcoded adapters and barcode blockers to match.
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 firstname.lastname@example.org 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.