Reference assisted nucleic acid sequence reconstruction from mass spectrometry data

While tandem MS has long been the main technique used for protein and small molecule identification in proteomics and metabolomics, MS-based protocols for nucleic acid analysis have only gained acceptance in the past decade. Commercially available from Sequenom, assays such as MassCLEAVE start by PCR amplification of one or more regions of interest using primers tagged with two different promoters (T7 and SP6). PCR amplification is followed by four in vitro transcription and RNA cleavage reactions that generate molecules corresponding to fragments ending at each occurrence of specific nucleotides in the original DNA template. Subjecting these fragments to matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS results in four base-specific mass spectra. Depending on instrument precision, peak masses can be matched with one or more fragment base compositions, or compomers. This information can be used for performing a number of nucleic acids analyses ranging from polymorphism discovery and genotyping and microbial identification to DNA methylation analysis and non-invasive prenatal genetic testing. MS-based assays are also becoming increasingly popular in molecular epidemiology due to the very low cost and relatively high throughput (384 reactions in less than one hour for a single MassARRAY system) compared to next-generation sequencing. They are a particularly good fit for studying heterogeneity of viral populations, since virus genomes are small and have even smaller hypervariable regions of common interest. For example, most studies of the Hepatitis C Virus (HCV) focus on a single viral amplicon containing the ≈290bp long Hypervariable region 1 (HVR1). This region is sufficient for estimating several population genetics parameters of interest. Analysis of genetic heterogeneity of hepatitis viruses is useful for tracing the route of transmission and the geographical migration of hepatitis carriers [1] and is essential for outbreak analys- s [2] and differentiation between acute and chronic forms of the disease [3].