Computational protocol: Draft Genome Sequence of Sphingobium quisquiliarum Strain P25T, a Novel Hexachlorocyclohexane (HCH) Degrading Bacterium Isolated from an HCH Dumpsite

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Protocol publication

[…] The disposal of hexachlorocylohexane (HCH) waste in the past has resulted in the pangenomic enrichment of various sphingomonad genotypes at HCH dumpsites (, ). In order to continue our efforts to sequence genomes of sphingomonads from the HCH dumpsite located near Lucknow, India (27°00′N and 81°09′E) (, ), we sequenced the genome of another sphingomonad strain, P25T (4.2 Mb).The draft genome sequence of strain P25T was obtained by use of an Illumina Genome Analyzer II platform. The sequencing data (n = 3,882,670; 90 bp/read) were assembled into contigs (n = 181, >500 bp) using ABySS 1.3.3 () set at a k-mer size of 47. Contigs (N50, 45 kb) were further validated (paired-end criterion) using bwa-0.5.9 (). Glimmer-3.02 () was used to predict the protein-encoding genes, whereas tRNA and rRNA genes were identified using ARAGORN () and RNAmmer (), respectively. A total of 4,033 coding sequences (CDS), 70 pseudogenes, 54 tRNA genes, and 1 rRNA operon were observed, with an average G+C content of 64%. Validated (paired-end criterion) genome assembly was annotated using RAST version 4.0 () and the NCBI Prokaryotic Genomes Automatic Annotation pipeline (PGAAP) ( Average nucleotide identity (ANI) () analysis revealed that Sphingobium japonicum UT26S (83.3%) (), Sphingobium indicum B90A (83.0%) (), and Sphingomonas sp. SKA58 (80.8%) are the closest phylogenetic neighbors of S. quisquiliarum P25T.The mechanisms of acquisition of lin genes in sphingomonads under HCH stress at these dumpsites are still not clearly understood (). The lin genes were first reported in S. japonicum UT26 () and subsequently from S. indicum B90A (). Many more sphingomonads have been isolated recently from the HCH dumpsite (, ). All of these strains by and large share the same pathway for the degradation of HCH isomers that requires the linA through linF genes (). Interestingly, the analysis of the draft genome of strain P25T revealed the presence of one copy each of linA, linH, linK, linL, linM, linN, and linX, and the IS FINDER database () ( predicted the occurrence of IS6 (n = 21), IS1380 (n = 4), IS3 (n = 1), and IS256 (n = 1) as the major transposon families. However, linB, which encodes haloalkane dehalogenase, was absent, indicating that this strain has yet to acquire linB through horizontal gene transfer.In comparison with the whole-genome sequence of S. japonicum UT26 (), P25T showed the presence of phenol- and toluene-degrading gene clusters, whereas homogentisate-, chlorophenol-, and anthranilate-degrading pathways were clearly absent in S. quisquiliarum P25T. Reciprocal smallest distance (RSD) analysis (e value, 10–15; distance, 0.125) revealed that S. quisquiliarum P25T and S. japonicum UT26 share 1,650 orthologous genes. Data and information assimilation from the complete genome of this species and a comparative analysis with other sphingomonad genomes () are under way to expand our understanding of HCH degradation, especially the rapid evolution and acquisition of lin genes in sphingomonads under HCH selection pressure. […]

Pipeline specifications

Software tools ABySS, BWA, Glimmer, ARAGORN, RNAmmer, RAST, PGAP
Applications Genome annotation, Phylogenetics, WGS analysis
Chemicals Lindane