PBHoover and CigarRoller: a method for confident haploid variant calling on Pacific Biosciences data and its application to heterogeneous population analysis

Ramirez-Busby, Sarah M; Elghraoui, Afif; Yb, Kim; K, Kim; Valafar, Faramarz

doi:10.1101/360370

Cited by 6 publications

(7 citation statements)

References 33 publications

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“…Raw Pacific Biosciences SMRT sequencing reads were aligned to the reference genome of the M. tuberculosis virulent type strain H37Rv reference strain (GenBank accession number NC_000962.3 ) using BLASR ( 25 ) (v1.3) with default parameters. PBHoover ( 26 ) ( https://gitlab.com/LPCDRP/pbhoover ) corrected aligned reads and called variants based on a maximum-likelihood criterion. VCF formatted files were further annotated with Variant Effect Predictor ( 27 ) (VEP) (v87) to determine the consequence of each variant.…”

Section: Methodsmentioning

confidence: 99%

Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis

Modlin

Marbach

Werngren

et al. 2021

Antimicrob Agents Chemother

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Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in M. tuberculosis strains with existing resistance to isoniazid and rifampicin (MDR) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, Pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotype pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of nineteen phenotypically PZA mono-resistant isolates collected in Sweden and compare their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDRPZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p-138) and N-terminal (ClpC1Val63Ala). Mutations have emerged in both these regions under POA selection in vitro and ClpC1N-terminal has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1Val63Ala mutants spanned 4 Indo-oceanic sublineages. Indo-oceanic isolates invariably harbored ClpC1Val63Ala and were starkly overrepresented (OR=22.2, p <0.00001) among PZA mono-resistant isolates (11/19) compared to MDRPZA-R isolates (5/80). The genetic basis of Indo-oceanic isolates’ overrepresentation in PZA mono-resistant TB remains undetermined, but substantial circumstantial evidence suggests ClpC1Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.

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Section: Methodsmentioning

confidence: 99%

Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis

Modlin

Marbach

Werngren

et al. 2021

Antimicrob Agents Chemother

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“…At the nucleotide level, the SNP rpoB :I1106T (T3317C) extended a homopolymer (from ATCCCG to ACCCCG). The 20 isolates with rpoB :I1106X were SMRT sequenced with P4C2 chemistry and variant called with PBHoover 25 , while the two isolates with rpoB :I1106T were SMRT sequenced with P6C4 chemistry and variant called with mummer 26 after de novo assembly.…”

Section: Resultsmentioning

confidence: 99%

“…For PacBio sequences, raw reads were aligned to M. tuberculosis H37Rv reference strain (Genbank accession number NC_000962.3) utilizing SMRT Analysis’ Basic Local Alignment with Successive Refinement (BLASR) with default parameters (v1.3) 38 . For variant calling, a custom software— PBHoover 25 (manuscript submitted)—corrected aligned reads and called variants based on a maximum likelihood criterion.…”

Section: Methodsmentioning

confidence: 99%

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Novel and Reported Compensatory Mutations in rpoABC Associate Specifically with Predominant Mycobacterium tuberculosis Rifampicin Resistance Marker rpoB:S450L

Conkle-Gutierrez

Ramirez-Busby

Gorman

et al. 2022

Preprint

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Background: Rifampicin (RIF) is a key first-line drug used to treat tuberculosis, a pulmonary disease caused by Mycobacterium tuberculosis. However antibiotic resistance to RIF is prevalent despite an apparent fitness cost. RIF resistance is primarily caused by mutations in the RIF resistance determining region in the rpoB gene, at the cost of slower growth in rich media. Compensatory mutations in the genes rpoA and rpoC have been shown to alleviate this fitness cost. These compensatory mutations may explain how RIF resistant strains have spread so rapidly. However, the effect of compensation on transmission is still unclear, partly because of uncertainty over which rpoABC mutations compensate for which RIF resistance markers. Objectives: We performed an association study on a globally representative set of 4309 whole genome sequenced clinical M. tuberculosis isolates to identify novel putative compensatory mutations, determine the prevalence of known and previously reported putative compensatory mutations, and determine which RIF resistance markers associate with these compensatory mutations. Results and Conclusions: Only 20.0% (216/1079) of RIF resistant isolates carried previously reported high-probability compensatory mutations, suggesting existence of other compensatory mutations. Using a strict phylogenetic approach, we identified 18 novel putative compensatory mutations in rpoC, rpoB, and rpoA. Novel and previously reported compensatory mutations were strongly associated with the RIFR marker rpoB:S450L, suggesting compensation may be specific to particular RIFR markers. These findings will aid identification of RIF-resistant M. tuberculosis strains with restored fitness. Such strains pose a greater risk of causing resistant outbreaks.

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“…For 268 isolates sequenced on the PacBio RSI platform, raw reads were aligned to M. tuberculosis H37Rv reference strain (Genbank accession number NC_000962.3) utilizing SMRT Analysis’ BLASR with default parameters (v1.3) ( Chaisson and Tesler, 2012 ). For variant calling, a custom software, PBHoover ( Ramirez-Busby et al, 2018 ) (manuscript submitted), corrected aligned reads and called variants based on a maximum likelihood criterion. PBHoover determines the threshold number of supporting reads for each variant based on the sequencing depth, the type of mutation, and the per base per read for the specific mutation type and sequencing platform ( Ramirez-Busby et al, 2018 ).…”

Section: Methodsmentioning

confidence: 99%

Novel and reported compensatory mutations in rpoABC genes found in drug resistant tuberculosis outbreaks

Conkle-Gutierrez,

Ramirez-Busby,

Gorman

et al. 2024

Front. Microbiol.

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BackgroundRifampicin (RIF) is a key first-line drug used to treat tuberculosis, a primarily pulmonary disease caused by Mycobacterium tuberculosis. RIF resistance is caused by mutations in rpoB, at the cost of slower growth and reduced transcription efficiency. Antibiotic resistance to RIF is prevalent despite this fitness cost. Compensatory mutations in rpoABC genes have been shown to alleviate the fitness cost of rpoB:S450L, explaining how RIF resistant strains harbor this mutation can spread so rapidly. Unfortunately, the full set of RIF compensatory mutations is still unknown, particularly those compensating for rarer RIF resistance mutations.ObjectivesWe performed an association study on a globally representative set of 4,309 whole genome sequenced clinical M. tuberculosis isolates to identify novel putative compensatory mutations, determine the prevalence of known and previously reported putative compensatory mutations, and determine which RIF resistance markers associate with these compensatory mutations.Results and conclusionsOf the 1,079 RIF resistant isolates, 638 carried previously reported putative and high-probability compensatory mutations. Our strict criteria identified 46 additional mutations in rpoABC for which no strong prior evidence of their compensatory role exists. Of these, 35 have previously been reported. As such, our independent corroboration adds to the mounting evidence that these 35 also carry a compensatory role. The remaining 11 are novel putative compensatory markers, reported here for the first time. Six of these 11 novel putative compensatory mutations had two or more mutation events. Most compensatory mutations appear to be specifically compensating for the fitness loss due to rpoB:S450L. However, an outbreak of 22 closely related isolates each carried three rpoB mutations, the rare RIFR markers D435G and L452P and the putative compensatory mutation I1106T. This suggests compensation may require specific combinations of rpoABC mutations. Here, we report only mutations that met our very strict criteria. It is highly likely that many additional rpoABC mutations compensate for rare resistance-causing mutations and therefore did not carry the statistical power to be reported here. These findings aid in the identification of RIF resistant M. tuberculosis strains with restored fitness, which pose a greater risk of causing resistant outbreaks.

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PBHoover and CigarRoller: a method for confident haploid variant calling on Pacific Biosciences data and its application to heterogeneous population analysis

Abstract: Motivation: Single Molecule Real-Time (SMRT) sequencing has important and underutilized

Cited by 6 publications

References 33 publications

Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis

Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis

Novel and Reported Compensatory Mutations in rpoABC Associate Specifically with Predominant Mycobacterium tuberculosis Rifampicin Resistance Marker rpoB:S450L

Novel and reported compensatory mutations in rpoABC genes found in drug resistant tuberculosis outbreaks

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