Sequencing of mpox fragments
After Qubit quantification using Qubit® dsDNA HS Assay Kit and Qubit 4.0
fluorometer (Thermo Fisher) amplicons were fragmented (Bioruptor,
Diagenaode) into fragments of 250 bp long. Libraries were built adding
barcode, for sample identification, and primers to fragmented DNA with
Ion Plus Fragment Library Kit
using AB Library Builder System (Thermo Fisher Scientific). To pool
equimolarly the barcoded samples, a quantification step by real time PCR
using Ion Library TaqMan™ Quantitation Kit (Thermo Fisher) was realized.
An emulsion PCR of the pools and loading on 530 Chip were
performed using the automated Ion Chef instrument (Thermo
Fisher). Sequencing was performed using the S5 XL Ion torrent technology
(Thermo Fisher) following manufacturer’s instructions. Consensus
sequence was obtained after trimming of reads (reads with quality score
< 0.99, and length < 100 bp were removed and the 30
first and 30 last nucleotides were removed from the reads) and mapping
on the MT903344.1 mpox reference sequence using CLC genomics workbench
software v.21.0.5 (Qiagen). Parameters for reference-based assembly
consisted of match score = 1, mismatch cost = 2, length fraction = 0.5,
similarity fraction = 0.8, insertion cost = 3, and deletion cost = 3.
A de novo contig was also produced to ensure that the consensus
sequence was not affected by the reference sequence.