Pacific Biosciences of California Inc (PACB)

[Paulieme]

Complete Genome Sequence of Achromobacter xylosoxidans MN001, a
Cystic Fibrosis Airway Isolate--- (This work was supported by the University of Minnesota Medical School.
We thank Karl Oles (Mayo Clinic Bioinformatics Core) for performing
PacBio library preparation and sequencing, and Kevin Silverstein,
John Vestrum, and the staff at the UMN MSI for their efforts in installing
and supporting PacBio SMRT Analysis. We also thank the patients and
caregivers at the UMN Adult CF Treatment Center for their support of
this research.) Achromobacter xylosoxidans is an aerobic Gram-negative bacterium
that is widely distributed throughout freshwater and soil
environments. This bacterium is also an opportunistic human
pathogen of immunocompromised hosts and is commonly associated
with a range of respiratory infections. In individuals with
cystic fibrosis (CF), A. xylosoxidans has seen an increase in prevalence,
with some treatment centers reporting positive culture rates
as high as 17.6% (1). Culture-independent 16S rRNA gene studies
suggest that this frequency might be even higher. Despite its association
with chronic lung infections and poor pulmonary function
scores (2), the impact of A. xylosoxidans on CF disease progression
is not entirely clear. This uncertainty, in addition to the bacterium’s
multidrug resistance (3), robust biofilm formation (4), and
transmissibility (5), warrants further study of the molecular basis for
its phenotypes. Here, we report the complete genome sequence of
A. xylosoxidans MN001—a multidrug-resistant isolate recovered
from multiple patients at the University of Minnesota Cystic Fibrosis
Center (institutional review board approval 1401M47262).
Genomic DNA (gDNA) was isolated from MN001 using the
Wizard purification kit (Promega) and sequenced using singlemolecule
real-time (SMRT) and Illumina technologies. SMRT libraries
were constructed according to Pacific Biosciences protocols
with a 20-kb insert size. Following ligation of SMRTbell
adapters, sheared gDNA was size selected with a 4-kb cutoff using
Blue Pippin electrophoresis (Sage Science) to generate a greater
fraction of long reads capable of resolving repeat sequencing in the
A. xylosoxidans genome. Sequencing was performed using the
PacBio RS II platform. Subread filtering from 2 SMRTcells captured
with a 240-min movie and P6-C4 chemistry yielded 592 Mbp of sequence
reads with an average read length of 11,520 bp ( N50, 16,106
bp). Assembly was performed using the Hierarchical Genome Assembly
Process (HGAP) version 3 (6) in SMRT Analysis version 2.2
hosted on the University of Minnesota supercomputer. Remaining
indels were removed with three successive passes through Quiver to
achieve a final consensus accuracy of 99.9997% (QV 56) at 100
coverage. This assembly consisted of one circular contig representing
a 5,876,039-bp chromosome. Illumina libraries were analyzed using MiSeq with 250-bp paired-end sequences, yielding ~2.5 million
reads. Reads were mapped onto the SMRT-derived contig using
breseq version 0.24rc6 (7),andthe 10 indels remaining after polishing
were corrected using Pilon version 1.10 (8), yielding essentially perfect
final per-base accuracy. TheMN001genome was annotated with
Prokka version 1.11 (9) using A. xylosoxidans NH44874-1996 (4) as
the reference genome.
At 5.8 Mbp, the MN001 genome is remarkably smaller than the
seven A. xylosoxidans genomes published to date. The GC content
is 67.72%, which is consistent with previously sequenced
strains. A total of 5,328 genes, including 10 rRNA (3 16S, 3 23S, 4
5S) and 67 tRNA genes, were annotated. Function was assigned
for 4,333 of 5,251 predicted coding sequences (82%). Detailed
analyses of the MN001 genome, including comparative studies
with other A. xylosoxidans strains are in progress.
Nucleotide sequence accession numbers. The assembly and
annotation have been deposited in GenBank under the accession
number CP012046. PacBio and Illumina reads have been deposited
to the NCBI Sequence Read Archive under BioProject number
PRJNA288995.
ACKNOWLEDGMENTS
This work was supported by the University of Minnesota Medical School.
We thank Karl Oles (Mayo Clinic Bioinformatics Core) for performing
PacBio library preparation and sequencing, and Kevin Silverstein,
John Vestrum, and the staff at the UMN MSI for their efforts in installing
and supporting PacBio SMRT Analysis. We also thank the patients and
caregivers at the UMN Adult CF Treatment Center for their support of
this research.
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