Validation of high throughput sequencing and microbial forensics applications

Abstract

High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.

Keywords: Bioinformatics; High throughput sequencing; Library preparation; Microbial forensics; Sample preparation; Validation.

Figure 1

Basic schematic of data flow through an analysis process. The first step of base calling generally is completed by the instrument software, and each downstream step must be included in the validated analytical pipeline. Additional data processing after generating sequence reads is required, for example with contig building and/or alignment, and will depend on the application.

Figure 2

Alternate alignments of identical sequences. Reads 1 and 2 are aligned in equally optimal ways that indicate different locations for a 2 bp deletion relative to the reference. Differences in alignment can be problematic when an evidence sample’s consensus alignment is based on a different approach than that of the reference sample or entries in a database.