P096 : EVALUATION OF ENZYMATIC LIBRARY PREPARATION FOR THE ILLUMINA MISEQ

Abstract

Aim We have previously considered transposon-and sonication-based library preparation methods for next generation sequencing of HLA-A, B, C, DRB1 and DQB on the Illumina MiSeq, and found that they are not suitable for routine clinical work. Here, we evaluated reagents from NEB, NEBNext dsDNA Fragmentase and NEBNext DNA Library Prep Master Mix, to generate sequencing libraries for clinical testing. Methods We performed long-range PCR for HLA-A, B, C, DRB1 and DQB1 on two IHWG samples using a combination of in-house and commercial primers. The amplified products ranged from 3900 to 4800 bp in length. The class I primers were designed to cover all exons, introns and UTRs whereas the class II primers covered exon 2 to the 3′UTR. Samples were enzymatically fragmented using NEBNext dsDNA Fragmentase which is composed of two different endonucleases isolated from V. vulnificus and T7. Length of incubation time of the amplicon and Fragmentase mixture determines general fragment length. We compared the ability of the endonucleases to generate fragments of two lengths, 100–300 bp and 300–600 bp, respectively. Following fragmentation, we prepared libraries for sequencing with the NEBNext Library Prep kit. Unique indices were ligated following end repair and dA-tailing of the fragments. Fragment size was determined by the Agilent 2100 Bioanalyzer instrument. Samples were equimolarly pooled and ran on the Illumina MiSeq using Reagent kit v2 for both 300 and 500 cycle runs. Results The two Fragmentase incubation times suggested by the manufacturer produced two very different average fragment sizes. Including the adaptor and index, the average size of the 100–300 bp incubation time was 273 bp and the 300–600 bp incubation time yielded a product of 619 bp. Conclusions Use of the NEBNext dsDNA Fragmentase allows the generation of fragments of different sizes. Although we easily generated our desired fragment sizes, we found the NEBNext DNA Library Prep Master Kit to be extremely time- and labor intensive requiring numerous transfer steps. Additionally, this kit requires a large number of AMPure XP Beads for sample clean-up. Analyses are in progress to evaluate the effect that size and library preparation method has on HLA typing assignments.