Pacific Biosciences of California Inc (PACB)

[Paulieme]

Comparative genome analysis of Wolbachia strain
wAu /// Results and discussion
Genomic DNA purity assessment
Approximate calculations based on quantitative PCR (qPCR) C(t) values for wAu and host
genes were performed to estimate the degree of contamination with host gDNA in wAu
gDNA samples extracted from cultured cells and whole adult flies. The estimated purity of
wAu gDNA was ~60% for the extract from cultured cells, and >90% for the extract from
whole adult flies. The latter is comparable to the figure of up to 97% reported previously [27]
using the same extraction method. There is no previous data on Wolbachia gDNA extraction
from cultured cells. One explanation for the lower purity could is that Wolbachia densities
may be lower within cultured cells than in vivo.
Genome sequencing and assembly
wAu genome sequencing was initially performed using the Illumina platform on gDNA
extracted from whole adult files. However, the resulting assembly was fragmented in the
regions of most interest, with scaffold positions uncertain. A second round of sequencing was
therefore performed using the PacBio RS II system to obtain longer reads in an attempt to
improve the assembly, using gDNA extracted from cultured cells rather whole adult files.
The Illumina data was used to correct errors in the PacBio reads, which assembled into a
single contig.
The achievement of a single contig assembly shows that PacBio represents an extremely
useful new sequencing platform for rapid generation of finished bacterial genome assemblies.
Furthermore, the generation of this single contig from a very small amount of DNA
(approximately 2 ng), containing a substantial amount of host DNA contamination (~40%),
suggests that PacBio is well suited to use in cases where it is hard to obtain a large amount of
gDNA, including obligate endosymbionts, like Wolbachia, that cannot be cultured outside of
host cells. The sequence generated was largely consistent with data produced using the
Illumina platform, with only one single nucleotide polymorphism (SNP) between the two
datasets. There were 88 indels relative to Illumina data; these were mostly single nucleotide,Conclusions
In this study, a methodology for conveniently extracting Wolbachia gDNA for genome
sequencing using an infected cell line has been successfully employed, and the PacBio RS II
sequencing platform has proved a very useful tool for achieving a complete bacterial
assembly, particularly when combined with Illumina sequencing. Using this approach, a
single contig assembly has been generated for the genome of the wAu strain, which does not
induce CI. Comparison of this genome to that of wMel, which does induce CI, revealed
significant structural differences in the prophage regions and loss or potential inactivation of
a number of genes.(click on link for lots more,28 pages) http://www.biomedcentral.com/content/pdf/1471-2164-15-928.pdf