Abstract
Next generation sequencing and bioinformatic approaches are increasingly used to quantify microorganisms within populations by analysis of ‘meta-barcode’ data. This approach relies on comparison of amplicon sequences of ‘barcode’ regions from a population with public-domain databases of reference sequences. However, for many organisms relevant ‘barcode’ regions may not have been identified and large databases of reference sequences may not be available. A workflow and software pipeline, ‘MetaGaAP,’ was developed to identify and quantify genotypes through four steps: shotgun sequencing and identification of polymorphisms in a metapopulation to identify custom ‘barcode’ regions of less than 30 polymorphisms within the span of a single ‘read’, amplification and sequencing of the ‘barcode’, generation of a custom database of polymorphisms, and quantitation of the relative abundance of genotypes. The pipeline and workflow were validated in a ‘wild type’ Alphabaculovirus isolate, Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV-AC53) and a tissue-culture derived strain (HaSNPV-AC53-T2). The approach was validated by comparison of polymorphisms in amplicons and shotgun data, and by comparison of predicted dominant and co-dominant genotypes with Sanger sequences. The computational power required to generate and search the database effectively limits the number of polymorphisms that can be included in a barcode to 30 or less. The approach can be used in quantitative analysis of the ecology and pathology of non-model organisms.
Highlights
Culture-independent molecular techniques to identify and quantify components of microbial communities have been facilitated by the use of generation sequencing (NGS) [1,2].Shotgun sequencing and whole or partial genome assembly uses algorithms comparing sequence data to public sequence databases [2,3,4,5,6]
Amplicon sequencing introduces bias resulting from gene copy number, selection of primers, and classification based on limited span of Biology 2017, 6, 14; doi:10.3390/biology6010014
The baculovirus isolate HaSNPV-AC53 was obtained from AgBiTech Pty Ltd., passaged once in
Summary
Culture-independent molecular techniques to identify and quantify components of microbial communities have been facilitated by the use of generation sequencing (NGS) [1,2].Shotgun sequencing and whole or partial genome assembly uses algorithms comparing sequence data to public sequence databases (such as Genbank) [2,3,4,5,6]. Of fungi or cytochrome oxidase) and comparison to sequence databases specific to those regions to determine taxonomic assignment and relative abundance of taxa in the community [2,7,8,9,10,11]. Both approaches are limited by available sequencing technology that relies on partial genome ‘reads’, and by the scope and accuracy of sequences in the reference databases. Amplicon sequencing introduces bias resulting from gene copy number, selection of primers, and classification based on limited span of Biology 2017, 6, 14; doi:10.3390/biology6010014 www.mdpi.com/journal/biology
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