title: SISPA-Seq for rapid whole genome surveys of bacterial isolates.
abstract: Whole genome sequencing (WGS) of large isolate collections has many applications, yet sequencing costs are still significant. We sought to develop a rapid and cost efficient WGS method to address fundamental questions in clinical microbiology. We evaluated the performance of SISPA (Sequence-Independent, Single-Primer Amplification) combined with next-generation sequencing (SISPA-Seq) of 75 clinical isolates of Acinetobacter baumannii to establish whether SISPA-Seq resulted in sufficient coverage and quality to (1) determine strain phylogenetic placement and (2) and carriage of known antibiotic resistance (AbR) genes. Strains for which whole genome sequences were available were included for validation. Two libraries for each strain were constructed from separate SISPA reactions with different barcoded primers, using genomic DNA prepared from either high quality or rapid heat-lysis preparations. SISPA-Seq resulted in a median of 65× genome coverage when reads from both primer sets were combined. Coverage and quality were sufficient for detection of AbR genes by comparison of reads to the ARG-ANNOT database and were often sufficient to distinguish between different allelic variants of the same gene. kSNP and RAxML were used to construct a robust phylogeny based on single-nucleotide variants (SNVs) that showed that the SISPA-Seq data was sufficient for sensitive and accurate phylogenetic placement. Advantages of the SISPA-Seq method include inexpensive and rapid DNA preparation and a typical total cost less than one-half that of standard genome sequencing. In summary, SISPA-Seq can be used to survey whole genomes of a large strain collection and identify strains that should be targeted for additional sequencing.
authors: J. Craig Venter Institute, La Jolla, CA, USA.
authors: Meredith S
authors: MS
authors: Wright
authors: J. Craig Venter Institute, Rockville, MD, USA.
authors: Timothy B
authors: TB
authors: Stockwell
authors: J. Craig Venter Institute, Rockville, MD, USA.
authors: Erin
authors: E
authors: Beck
authors: J. Craig Venter Institute, Rockville, MD, USA.
authors: Dana A
authors: DA
authors: Busam
authors: University Hospitals Case Medical Center, USA.
authors: Saralee
authors: S
authors: Bajaksouzian
authors: University Hospitals Case Medical Center, USA; Case Western Reserve University, USA.
authors: Michael R
authors: MR
authors: Jacobs
authors: Case Western Reserve University, USA; Louis Stokes Cleveland Veterans Affairs Medical Center, USA.
authors: Robert A
authors: RA
authors: Bonomo
authors: J. Craig Venter Institute, La Jolla, CA, USA. Electronic address: madams@jcvi.org.
authors: Mark D
authors: MD
authors: Adams
mesh_terms: D000151:Acinetobacter Infections / Q000188:drug therapy / Q000382:microbiology; D040981:Acinetobacter baumannii / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D010802:Phylogeny; D020641:Polymorphism, Single Nucleotide / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial
keywords: Acinetobacter baumannii; SISPA; SISPA-Seq; Whole genome survey