Whole Genome Sequencing of MDR Strains of Pseudomonas aeruginosa and Mycobacterium tuberculosis Using the “Ion Torrent Personal Genome Machine”

The introduction of molecular detection technique namely the Sanger sequencing in the year 1977 made sequencing the order of nucleotides in a DNA molecule possible. It made a vast difference in the field of biology as the sequence abnormality detection in genetic disorders became much easier. In the year 1983, PCR technique was developed. And, application of it in a wide field of biology was easier, could be automated and required a lesser amount of analytic material. Application of PCR was preferred also in the field of ocular microbiology due to its advantage over the conventional microbiology. Over the years, PCR-based sequencing technique was developed by ABI-applied biology system wherein sequencing of short fragments of DNA became much easier. Human genome was sequenced with overlapping primer sequences amplified PCR products run in DNA sequences. This was called primer walking. The disadvantages involved in PCR technique led to the development of next-generation sequencers NGS wherein a large fragment/whole genome sequencing of an organism became possible. One sharp contrast to the first-generation Sanger sequencing is that NGS generates short reads of frequently less than 500 bp as opposed to over 1000 bp. However, the massive depth of coverage, i.e. multiple reads over the same template DNA region, compensates for the limitations of short reads. The NGS technologies have drastically increased the speed and throughput capacities over the Sanger sequencing while reducing cost, even as we write. One sharp contrast to the firstgeneration Sanger sequencing is that NGS generates short reads of frequently less than 500 bp as opposed to over 1000 bp. However, the massive depth of coverage, i.e. multiple reads over the same template DNA region, compensates for the limitations of short reads. In the field of microbiology, NGS has added knowledge in knowing total microbiome of human site which otherwise would have taken the scientist through a painstaking path. Many kits are available wherein 16S RNA– based primers are used to simultaneously apply the entire microbial flora and pathogen from a clinical sample. Upon completion of sequencing using any of the second NGS platforms, the data are analysed to know the various organisms present in it. In any of the NGS techniques, the real challenge is the data analysis. The amount of data output should be analysed appropriately. In L&T Microbiology Research Centre, two research projects were conducted with the research grant from ICMR, New Delhi, for whole genome sequencing of multidrug-resistant (MDR) strains of Pseudomonas aeruginosa and the other grant from Chennai Willingdon Corporate Foundation (CWCF), Chennai, for MDR Mycobacterium tuberculosis (MDR-TB) isolates from Chennai population.