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KARGA: Multi-platform Toolkit for k-mer-based Antibiotic Resistance Gene Analysis of High-throughput Sequencing Data.

ABSTRACT: High-throughput sequencing is widely used for strain detection and characterization of antibiotic resistance in microbial metagenomic samples. Current analytical tools use curated antibiotic resistance gene (ARG) databases to classify individual sequencing reads or assembled contigs. However, identifying ARGs from raw read data can be time consuming (especially if assembly or alignment is required) and challenging, due to genome rearrangements and mutations. Here, we present the k-mer-based antibiotic gene resistance analyzer (KARGA), a multi-platform Java toolkit for identifying ARGs from metagenomic short read data. KARGA does not perform alignment; it uses an efficient double-lookup strategy, statistical filtering on false positives, and provides individual read classification as well as covering of the database resistome. On simulated data, KARGA's antibiotic resistance class recall is 99.89% for error/mutation rates within 10%, and of 83.37% for error/mutation rates between 10% and 25%, while it is 99.92% on ARGs with rearrangements. On empirical data, KARGA provides higher hit score (≥1.5-fold) than AMRPlusPlus, DeepARG, and MetaMARC. KARGA has also faster runtimes than all other tools (2x faster than AMRPlusPlus, 7x than DeepARG, and over 100x than MetaMARC). KARGA is available under the MIT license at https://github.com/DataIntellSystLab/KARGA.

SUBMITTER: Prosperi M

PROVIDER: S-EPMC8383893 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Comprehensive microbial risk assessment requires high-throughput quantification of diverse microbial risks in the environment. Current metagenomic next-generation sequencing approaches can achieve high-throughput detection of genes indicative of microbial risks but lack quantitative capabilities. This study developed and tested a quantitative metagenomic next-generation sequencing (qmNGS) approach. Numerous xenobiotic synthetic internal DNA standards were used to determine the sequencing yield (Yseq) of the qmNGS approach, which can then be used to calculate absolute concentration of target genes in environmental samples based on metagenomic sequencing results. The qmNGS approach exhibited excellent linearity as indicated by a strong linear correlation (r2 = 0.98) between spiked and detected concentrations of internal standards. High-throughput capability of the qmNGS approach was demonstrated with artificial Escherichia coli mixtures and cattle manure samples, for which 95 ± 3 and 208 ± 4 types of antibiotic resistance genes (ARGs) were detected and quantified simultaneously. The qmNGS approach was further compared with quantitative real-time PCR (qPCR) and demonstrated comparable levels of accuracy and less variation for the quantification of six target genes (16S, tetO, sulI, tetM, ermB, and qnrS). IMPORTANCE Monitoring and comprehensive assessment of microbial risks in the environment require high-throughput gene quantification. The quantitative metagenomic NGS (qmNGS) approach developed in this study incorporated numerous xenobiotic and synthetic DNA internal standard fragments into metagenomic NGS workflow, which are used to determine a new parameter called sequencing yield that relates sequence base reads to absolute concentration of target genes in the environmental samples. The qmNGS approach demonstrated excellent method linearity and comparable performance as the qPCR approach with high-throughput capability. This new qmNGS approach can achieve high-throughput and accurate gene quantification in environmental samples and has the potential to become a useful tool in monitoring and comprehensively assessing microbial risks in the environment.

Dataset Information

KARGA: Multi-platform Toolkit for k-mer-based Antibiotic Resistance Gene Analysis of High-throughput Sequencing Data.

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