June 1, 2021  |  

A comparison of 454 GS FLX Ti and PacBio RS in the context of characterizing HIV-1 intra-host diversity.

PacBio 2013 User Group Meeting Presentation Slides: Lance Hepler from UC San Diego’s Center for AIDS Research used the PacBio RS to study intra-host diversity in HIV-1. He compared PacBio’s performance to that of 454® sequencer, the platform he and his team previously used. Hepler noted that in general, there was strong agreement between the platforms; where results differed, he said that PacBio data had significantly better reproducibility and accuracy. “PacBio does not suffer from local coverage loss post-processing, whereas 454 has homopolymer problems,” he noted. Hepler said they are moving away from using 454 in favor of the PacBio system.


June 1, 2021  |  

Using whole exome sequencing and bacterial pathogen sequencing to investigate the genetic basis of pulmonary non-tuberculous mycobacterial infections.

Pulmonary non-tuberculous mycobacterial (PNTM) infections occur in patients with chronic lung disease, but also in a distinct group of elderly women without lung defects who share a common body morphology: tall and lean with scoliosis, pectus excavatum, and mitral valve prolapse. In order to characterize the human host susceptibility to PNTM, we performed whole exome sequencing (WES) of 44 individuals in extended families of patients with active PNTM as well as 55 additional unrelated individuals with PNTM. This unique collection of familial cohorts in PNTM represents an important opportunity for a high yield search for genes that regulate mucosal immunity. An average of 58 million 100bp paired-end Illumina reads per exome were generated and mapped to the hg19 reference genome. Following variant detection and classification, we identified 58,422 potentially high-impact SNPs, 97.3% of which were missense mutations. Segregating variants using the family pedigrees as well as comparisons to the unrelated individuals identified multiple potential variants associated with PNTM. Validations of these candidate variants in a larger PNTM cohort are underway. In addition to WES, we sequenced the genomes of 52 mycobacterial isolates, including 9 from these PNTM patients, to integrate host PNTM susceptibility with mycobacterial genotypes and gain insights into the key factors involved in this devastating disease. These genomes were sequenced using a combination of 454, Illumina, and PacBio platforms and assembled using multiple genome assemblers. The resulting genome sequences were used to identify mycobacterial genotypes associated with virulence, invasion, and drug resistance.


June 1, 2021  |  

Resolving the ‘dark matter’ in genomes.

Second-generation sequencing has brought about tremendous insights into the genetic underpinnings of biology. However, there are many functionally important and medically relevant regions of genomes that are currently difficult or impossible to sequence, resulting in incomplete and fragmented views of genomes. Two main causes are (i) limitations to read DNA of extreme sequence content (GC-rich or AT-rich regions, low complexity sequence contexts) and (ii) insufficient read lengths which leave various forms of structural variation unresolved and result in mapping ambiguities.


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