DNA antibiotic resistance genes MTB sample 2
The targeted multiplex PCR covered 18 resistance genes and primer sets were multiplexed into 3 pools. Each primer pool contains genes corresponding to groups classified by the WHO that are based on their associated clinical drug of choice. Pool 1 target genes are associated with first-line drugs and, as control, a MTB diagnostic marker: rpoB, katG, inhA, fabG1, embB, pncA and IS6110. Pool 2 includes genes linked to second-line drugs from group A and B: gyrA, gyrB, atpE, Rv0678, pepQ, rplC and rrl. Pool 3 consists of genes associated with group C drugs, including ddn, rrs, rpsL, gid and ethA. For PCR reactions the Q5 High-Fidelity DNA Polymerase (New Engeland Biolabs, Ipswich, MA) was used in a total reaction volume of 25 L, according to the manufacturers protocol. PCR reaction settings were as follows: pre-denaturation at 98C for 30s, followed by 39 cycles of denaturation at 98C for 10s, annealing at 69C for 30s, extension at 72C for 1min, and a final extension at 72C for 2min. DNA concentrations were measured with the FLUOstar OMEGA Microplate Reader (BMG Labtech, Ortenberg, Germany). Sequencing reactions were prepared using the Native Barcoding Kit 96 V14 (SQK-NBD114.96) (Oxford Nanopore Technologies (ONT; Oxford, United Kingdom)). Library was loaded on R10 flow cells (ONT) and sequenced on a MinION Mk1B (ONT) with super accuracy basecalling (SUP) for 6 hours. In order to upload sequences in the Sequence Read Archive (SRA) human reads were extracted with the Homo sapiens genome (GCA_000001405.29).