GSM5464168: PRDM9 Amplicon Sequencing Pacbio CCS; Homo sapiens; OTHER
LongAmp Taq 2X Master Mix (M0287) from New England Biolabs Inc. was used for PCR amplification. Post-amplification, samples were individually tested for successful amplification and low presence of polymerase slippage (presence of DNA laddering/smearing) by running on agarose gel electrophoresis. Samples were re-amplified if there was extensive DNA laddering/smearing by visualization. Based on the Genome Reference Consortium Human Build 38 (PRDM9 allele with 13 ZFs): the final amplified product was 1,899bp which contained the 1,092bp C2H2 ZF array, 670bp of upstream flanking sequence, and 137bp of downstream flanking sequence to the PRDM9 ZF array. Of note, the total length of the final amplified product varied based on the number of ZFs present in the PRDM9 allele. Samples were then pooled and prepared for multiplexing. We performed dual-barcoding in order to sequence all 758 multiplexed individuals. The first round of barcoding was done by adding unique DNA barcode sequences to the 5' end of the PCR primers, totaling eight primer pairs. After amplification and the addition of the first barcode, samples were pooled in groups of eight (each sample tagged with a separate barcode sequence) and subjected to a second round of barcoding. The second round of multiplexing was performed using the PCR Barcoding Expansion 1-96 kit (EXP-PBC096) from Oxford Nanopore Technologies (ONT), Inc. following the protocol detailed on their website (https://nanoporetech.com - PCR barcoding (96) amplicons). Sequencing libraries for Pacbio were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA) and sequenced using the PacBio Sequel II System to generate Circular Consensus Sequencing (CCS) PacBio reads. Sequencing was performed with a 0.5 hour pre-extension and 10 hour recording time and a second sequencing run was performed with a 2 hour pre-extension and 30 hour recording time.