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.

Keywords: Acinetobacter baumannii; SISPA; SISPA-Seq; Whole genome survey.

Figure 1. The SISPA-Seq method

A. Overview of SISPA-Seq with phylogenetic and gene content analysis pipeline. B. SISPA reaction details. C. General design of SISPA primers.

Figure 2. Sequence read distribution

Example comparison of mapped read density for UH7007 A) SISPA-Seq preparation individual barcoding primer A; B) primer B; and c) a dataset of combined reads from both primers; or D) standard Illumina library preparation (‘WGS’). Reads were mapped to A. baumannii ACICU (CP000863) using bwa. Mean read density (reads per 10kb) for UH7007 WGS was 9504 (standard deviation (SD): 2244), compared to 8106 (SD: 4000) for the SISPA-Seq combined primers.

Figure 3. Relationship between read coverage and the number of informative SNVs

A) Effect of varying the minimum percentage of genomes required to have a base call at each SNV position for that SNV locus to be included in the SNV dataset, i.e., varying the parameter in ‘m’ in kSNP. B) Relationship between genome coverage and number of missing SNV values (i.e., N’s) for each genome.

Figure 4. kSNP-based phylogeny of SISPA-Seq and publically available A. baumannii genomes

The presence of selected antibiotic resistance genes is shown. Primary clades identified in (Wright et al., 2014) are indicated by colored branches, and node labels A–E. Strain names are color-coded based on the hospital from which the strain originated in the integrated hospital system. Bootstrap values for nodes with >50% support are indicated.