IMPORTANCE: This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.
METHODS: Genomic DNA obtained from a 55 years old, self-declared healthy, anonymous male of Malay descent was sequenced. The subject's mother died of lung cancer and the father had a history of schizophrenia and deceased at the age of 65 years old. A systematic, intuitive computational workflow/pipeline integrating custom algorithm in tandem with large datasets of variant annotations and gene functions for genetic variations with pharmacogenomics impact was developed. A comprehensive pathway map of drug transport, metabolism and action was used as a template to map non-synonymous variations with potential functional consequences.
PRINCIPAL FINDINGS: Over 3 million known variations and 100,898 novel variations in the Malay genome were identified. Further in-depth pharmacogenetics analysis revealed a total of 607 unique variants in 563 proteins, with the eventual identification of 4 drug transport genes, 2 drug metabolizing enzyme genes and 33 target genes harboring deleterious SNVs involved in pharmacological pathways, which could have a potential role in clinical settings.
CONCLUSIONS: The current study successfully unravels the potential of personal genome sequencing in understanding the functionally relevant variations with potential influence on drug transport, metabolism and differential therapeutic outcomes. These will be essential for realizing personalized medicine through the use of comprehensive computational pipeline for systematic data mining and analysis.
Methods: Two complementary approaches, saturated transposon mutagenesis and spontaneous mutation induction with high concentrations of colistin and polymyxin B, were employed to select for mutations associated with resistance to polymyxins. Mutants were identified using transposon-directed insertion-site sequencing or Illumina WGS. A resistance phenotype was confirmed by MIC and further investigated using RT-PCR. Competitive growth assays were used to measure fitness cost.
Results: A transposon insertion at nucleotide 41 of the pmrB gene (EC958pmrB41-Tn5) enhanced its transcript level, resulting in a 64- and 32-fold increased MIC of colistin and polymyxin B, respectively. Three spontaneous mutations, also located within the pmrB gene, conferred resistance to both colistin and polymyxin B with a corresponding increase in transcription of the pmrCAB genes. All three mutations incurred a fitness cost in the absence of colistin and polymyxin B.
Conclusions: This study identified the pmrB gene as the main chromosomal target for induction of colistin and polymyxin B resistance in E. coli.
FINDINGS: We optimized the assembly of a Hevea bark transcriptome based on 16 Gb Illumina PE RNA-Seq reads using the Oases assembler across a range of k-mer sizes. We then assessed assembly quality based on transcript N50 length and transcript mapping statistics in relation to (a) known Hevea cDNAs with complete open reading frames, (b) a set of core eukaryotic genes and (c) Hevea genome scaffolds. This was followed by a systematic transcript mapping process where sub-assemblies from a series of incremental amounts of bark transcripts were aligned to transcripts from the entire bark transcriptome assembly. The exercise served to relate read amounts to the degree of transcript mapping level, the latter being an indicator of the coverage of gene transcripts expressed in the sample. As read amounts or datasize increased toward 16 Gb, the number of transcripts mapped to the entire bark assembly approached saturation. A colour matrix was subsequently generated to illustrate sequencing depth requirement in relation to the degree of coverage of total sample transcripts.
CONCLUSIONS: We devised a procedure, the "transcript mapping saturation test", to estimate the amount of RNA-Seq reads needed for deep coverage of transcriptomes. For Hevea de novo assembly, we propose generating between 5-8 Gb reads, whereby around 90% transcript coverage could be achieved with optimized k-mers and transcript N50 length. The principle behind this methodology may also be applied to other non-model plants, or with reads from other second generation sequencing platforms.