Fig. 4

Application of Nextera DNA Flex to human amplicons. a Human leukocyte antigen (HLA) gene amplicons used as inputs for library preparation visualized on a 1% agarose gel. Lanes and expected amplicon sizes are as follows: 1, KBL Ladder; 2, HLA-A (4.1 kb); 3, HLA-B (2.8 kb); 4, HLA-C (4.2 kb); 5, HLA-DPA1 (10.3 kb); 6, HLA-DPB1 (9.7 kb); 7, HLA-DQA1 (7.3 kb); 8, HLA-DRB2 (4.6 kb); 9, HLA-DQB1 (7.1 kb). b Nextera DNA Flex library yields of all HLA amplicons were within the acceptable values of > 4 ng/μl and 9–13 ng/μl for 1 ng and 100–300 ng inputs, respectively. The yields for Nextera DNA Flex libraries were higher than for those prepared using TruSight HLA; for TruSight HLA, libraries were prepared from 1 ng of each amplicon and then pooled. c The Bioanalyzer profiles depict library fragment size distributions within the acceptable range; the distribution is narrower for the Nextera DNA Flex libraries (1 ng DNA inputs) than the TruSight HLA libraries. d Sequencing coverage depth and uniformity were higher for libraries prepared using Nextera DNA Flex (Flex) compared with TruSight HLA (TS HLA). e Libraries were sequenced on a NextSeq 550, with downsampling to 25,000 reads per amplicon. Library preparation using Nextera DNA Flex (orange) resulted in more uniform coverage of the entire human mitochondrial chromosome when compared with Nextera XT (grey). The location of the PCR primers used to create the two mtDNA amplicons are depicted by blue and red arrows. Dotted-line rectangle indicates the D-Loop region. f Zoomed in view shows more uniform coverage with Nextera DNA Flex within the D-Loop region