DRMs detected at three thresholds by NGS are reported: 2%, 5% and 20% of the viral populace (the latter comparable to the detection threshold for Sanger sequencing).[11,18] Statistical methods If two or more mutations were present in the analysis of drug class-specific TDR by detection threshold (Fig. 9.2%/5.6%/3.2% for nucleoside reverse transcriptase inhibitors (NRTIs), 9.2%/6.6%/4.9% for non-NRTIs, 11.4%/5.5%/2.4% for protease inhibitors (PIs) and 3.5%/1.6%/0.1% for INSTI DRMs and varied by geographic region. Using the 2% detection threshold, individual DRMs with the highest prevalence were: PI M46IL (5.5%), RT K103NS (3.5%), RT G190ASE (3.1%), T215ISCDVEN (2.5%), RT M41L (2.2%), RT K219QENR (1.7%) and PI D30N (1.6%). INSTI DRMs were recognized almost specifically below the 20% detection threshold, most commonly Y143H (0.4%), Q148R (0.4%) and T66I (0.4%). Conclusions Use of NGS with this study populace resulted in the detection of a large proportion of low-level variants which would not have been recognized by traditional Sanger sequencing. Global monitoring studies utilizing NGS should provide a more comprehensive assessment of TDR prevalence in different regions of the world. for 15 min. The supernatant was extracted and centrifuged at 21 000 for 75 min and 360 L of the top supernatant was discarded. Viral RNA was extracted using QIAamp viral RNA extraction kit (Qiagen, Hilden, Germany) on a QIAcube robot using the manufacturers guidelines. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify two amplicons from your viral RNA. The primer 4E1RCat sequences (available in Table S5) were designed by Gall gene from amino acid position 1 to 935 where positions TM4SF18 1C99 encode protease (PR) protein, positions 100C659 encode RT protein, and positions 660C935 partially encode integrase (IN) protein (our amplicon did not cover position 936C947). Definition of transmitted drug resistance and phenotypic drug susceptibility As with a earlier paper from START reporting the results of locally performed Sanger sequencing, TDR was based on the WHO 2009 monitoring list with the help of RT mutations T215N and E138K [13,19]. INSTI mutations, which are not included on this list, were defined as those within the Stanford HIVdb monitoring DRM list, namely T66AIK, E92Q, F121Y, G140ACS, Y143CHR, S147G, Q148HKR and N155HS [20]. Interpretation of phenotypic drug susceptibility was standardized using the Stanford HIVdb algorithm v.8.6 which defines drug resistance as none, potential low level, low level, intermediate or high [21]. To achieve regularity with WHO resistance reports, expected potential low level is not reported. It is noted the Stanford HIVdb algorithm considers a much wider range of mutations than regarded as from the WHO 2009 monitoring list (including the integrase gene) and these additional TDR DRMs recognized by NGS were included for expected phenotypic drug susceptibility. Sequencing depth and thresholds for phoning DRMs Sequence go through coverage depth assorted markedly across the sequenced amplicons (highest in PR, intermediate in RT, least 4E1RCat expensive in IN). We stipulated a minimum go through depth of 200 across the region spanning all relevant mutations within each gene. For WHO monitoring mutations this comprised codons 23C90 of PR, codons 41C230 of RT, and codons 66C155 of IN; for Stanford expected phenotypic drug susceptibility this comprised codons 10C90 of PR, codons 41C348 of RT and codons 51C263 of IN. This resulted in different denominators for different drug classes, which were consequently analysed separately. DRMs recognized at three thresholds by NGS are reported: 2%, 5% and 20% of the viral populace (the latter comparable to the detection threshold for Sanger sequencing).[11,18] Statistical methods If two or 4E1RCat more mutations were present in the analysis of drug class-specific TDR by detection threshold (Fig. 4E1RCat 4E1RCat 1), the highest frequency was used in the analysis. Fishers exact test (two-sided) was used to test the association between geographical region and whether TDR variants were observed at 2C5%, 5C20% or 20%. Logistic regression analysis was.