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KAPA HyperPlex Adapters

Overview

Truncated adapters are compatible with ligation-based library construction workflows for direct and targeted multiplexed DNA and RNA (cDNA) sequencing applications on the Illumina® platform. Sequencing barcodes are added with UDI primer mixes during the library amplification step.

KAPA HyperPlex Adapters have a standard Illumina adapter backbone. They are therefore easily incorporated in new and existing Illumina sequencing workflows, and do not require customized sequencing primers. A combination of 8-nt sequencing barcodes on each of the i5 and i7 adapter oligos supports paired-end sequencing on 1- , 2- and 4-channel Illumina instruments. KAPA HyperPlex Adapters support a broad range of sample pooling (2- plex up to 384-plex, as appropriate for your workflow), for target enrichment and/or sequencing.

Unique dual-indexing is recommended for all applications and Illumina sequencers.1,2

Product highlights

  • Higher library complexity and fewer misassigned reads improve confidence in results
  • Unique dual-index combinations in KAPA HyperPlex Adapters allow reads with unexpected barcode combinations to be filtered out prior to data analysis
  • KAPA HyperPlex Adapters are manufactured using stringent procedures and undergo sequencing-based QC testing to reduce the potential for index misassignment resulting from barcode cross-contamination
  • Achieving the required library yield with less amplification cycles can increase library complexity and reduce PCR duplication
  • KAPA Universal Adapter is offered with or without UMI (Unique Molecular Indexes) for germline or somatic variant detection respectively


KAPA HyperPlex Adapters deliver high library yields in applications  that require library amplification
 

  • When input material is limited, robust library amplification delivers the maximum yield with the minimum number of cycles

  • The KAPA HyperPlex Adapters combine high library conversion rate with robust amplification efficiency

  • Achieving the required library yield with less amplification cycles can increase library complexity and reduce PCR duplication

Figure 1. Truncated adapters can deliver as much as almost twice the library yield generated by conventional full length adapters. Libraries were prepared  from 50 ng of human genomic DNA (NA12878; Coriell Institute) using the KAPA HyperPlus Kit and two different adapters. Twelve (n=12) DNA replicates were ligated to a  standard Illumina®-compatible full length Y-adapter and amplified for 7 cycles with standard Illumina-compatible library amplification primers. Another twelve (n=12) DNA  replicates were ligated to an Illumina-compatible truncated Y-adapter and amplified for 7 cycles with UDI Primer Mixes. All other conditions such as DNA fragmentation time,  final adapter, and primer concentration, as well as bead cleanup ratios were kept identical between the two sample sets. Libraries were quantified with a Qubit Fluorometer  (Thermo Fisher Scientific). The libraries prepared with a full length adapter had a mean yield of 1641.3 ng ±178 ng compared to a mean yield of 3119.2 ng ±157 ng for the  libraries prepared with the truncated adapter.

 

Somatic oncology research from cell-free DNA

  • Accurate molecule counting facilitated by molecular barcoded adapters is essential in somatic oncology applications,  especially from low inputs of cfDNA where every molecule counts

  • The KAPA Universal UMI Adapter has a proprietary design that prevents single errors in the UMI sequence to result in  false counting of spurious molecules

  • The KAPA Universal UMI Adapter shows improved performance in molecule counting, showing higher duplex recovery  and lower error rate than Supplier I’s UMI adapter

Figure 2. KAPA Universal UMI Adapter supports highly accurate molecule counting and high recovery of duplex molecules from 10 ng cfDNA. Five healthy donors’ cell-free DNA samples and the SeraSeq® ctDNA Complete™ Reference Material AF0.5% from SeraCare were tested in duplicate for library preparation and target enrichment with the  KAPA HyperCap Oncology Panel (214 Kb capture target). Libraries from 10 ng cfDNA were prepared with the KAPA HyperPrep Kit and captured according to the KAPA HyperCap cfDNA Workflow v1 for cfDNA in single hybridizations per sample. Sequencing clusters of 2 x 150 bp from a NextSeq™ 500 System were downsampled to 50 Million quality filtered clusters  per sample prior to  analysis.

High-performance – KAPA HyperCap Target Enrichment

  • Consistent high performing target enrichment workflows require extensive testing and optimization with complete reagent and kit  offering to achieve reproducible quality

  • KAPA Universal Adapter and KAPA UDI Primer Mixes have been validated with the KAPA HyperCap Workflow v3 and are compatible  with KAPA HyperPrep, KAPA HyperPlus and the KAPA RNA HyperPrep Kit (with or without RiboErase)

KAPA HyperPlex Adapter key sequencing metrics

Figure 3. KAPA Universal Adapter with all 384 KAPA UDI Primer Mixes perform consistently in the KAPA HyperCap Workflow v3. High reproducibility  was demonstrated when replicate libraries prepared with all 384 UDIs were pooled in the same sequencing run, delivering high specificity (% reads on-target), high uniformity  (% bases within 0.5x – 2x of median coverage) and deep target coverage (% bases covered by >50x). The KAPA HyperCap Heredity Panel (10 Mb capture target) was used  to enrich libraries which were prepared from 100 ng replicate inputs of human genomic DNA (NA12878; Coriell Institute) with the KAPA HyperPlus Kit in the KAPA HyperCap  Workflow v3. Pre-capture libraries were quantified with a Qubit Fluorometer (Thermo Fisher Scientific). An average pre-capture yield of 3.9 ±0.4 µg was obtained across the  384 libraries that were multiplexed by 12 in 32 hybridizations. All 384 final enriched libraries (32 captures) were sequenced on a NovaSeq™ 6000 System lane at 2 x 100 bp, resulting in a mean of ~28.4 Million clusters (56.8 M reads) per sample after quality filtering. After down-sampling to 10 Million clusters per sample, analysis followed the technical  note “How To Evaluate KAPA Target Enrichment Data” (March 2020). Total duplicate rate was 3.2 % ±0.2% and fold-80 base penalty was 1.32 ±0.01.

  1. https://www.illumina.com/science/education/minimizing-index-hopping.html. Accessed February 2019.
  2. Costello M, et al. BMC Genomics. 2018;19:332

Research Use Only. Not for use in diagnostic procedures.
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