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
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).
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 downsampling 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.