GenSeizer: a Multiplex PCR-Based Targeted Gene Sequencing Platform for Rapid and Accurate Identification of Major Mycobacterium Species.

Mycobacterium tuberculosis and nontuberculous mycobacterium (NTM) infections often exhibit similar clinical symptoms. Timely and effective treatment relies on the rapid and accurate identification of species and resistance genotypes. In this study, a new platform (GenSeizer), which combines bioinformatics analysis of a large data set and multiplex PCR-based targeted gene sequencing, was developed to identify 10 major Mycobacterium species that cause pulmonary, as well as extrapulmonary, human diseases. The simultaneous detection of certain erm(41) and rrl resistance genotypes in M. abscessus was also feasible. This platform was specific and sensitive and exhibited no cross-reactivity among reference strains and a detection limit of 5 DNA copies or 50 CFU Mycobacterium/ml. In a blind comparison, GenSeizer and multigene sequencing showed 100% agreement in the ability to identify 88 clinical Mycobacterium isolates. The resistance genotypes, confirmed by whole-genome sequencing of 30 M. abscessus strains, were also correctly identified by GenSeizer 100% of the time. These results indicate that GenSeizer is an efficient, reliable platform for detecting major pathogenic Mycobacterium species.

OTG-snpcaller: an optimized pipeline based on TMAP and GATK for SNP calling from ion torrent data.

Because the new Proton platform from Life Technologies produced markedly different data from those of the Illumina platform, the conventional Illumina data analysis pipeline could not be used directly. We developed an optimized SNP calling method using TMAP and GATK (OTG-snpcaller). This method combined our own optimized processes, Remove Duplicates According to AS Tag (RDAST) and Alignment Optimize Structure (AOS), together with TMAP and GATK, to call SNPs from Proton data. We sequenced four sets of exomes captured by Agilent SureSelect and NimbleGen SeqCap EZ Kit, using Life Technology's Ion Proton sequencer. Then we applied OTG-snpcaller and compared our results with the results from Torrent Variants Caller. The results indicated that OTG-snpcaller can reduce both false positive and false negative rates. Moreover, we compared our results with Illumina results generated by GATK best practices, and we found that the results of these two platforms were comparable. The good performance in variant calling using GATK best practices can be primarily attributed to the high quality of the Illumina sequences.