Palmer, J. M. | bioRxiv Science

bioRxiv2017

Non-biological synthetic spike-in controls and the AMPtk software pipeline improve fungal high throughput amplicon sequencing data

High throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal ITS amplicons, we created two ITS spike-in control mock communities composed of cloned DNA in plasmids: a biological mock community (BioMock), consisting of ITS sequences from fungal taxa, and a synthetic mock community (SynMock), consisting of non-biological ITS-like sequences. Using these spike-in controls we show that: 1) a non-biological synthetic control (e.g., SynMock) is the best solution for parameterizing bioinformatics pipelines, 2) pre-clustering steps for variable length amplicons are critically important, 3) a major source of bias is attributed to initial PCR reactions and thus HTAS read abundances are typically not representative of starting values. We developed AMPtk, a versatile software solution equipped to deal with variable length amplicons and quality filter HTAS data based on spike-in controls. While we describe herein a non-biological synthetic mock community for ITS sequences, the concept and AMPtk software can be widely applied to any HTAS dataset to improve data quality.\n\nAvailability and Implementation - AMPtk is publically available at https://github.com/nextgenusfs/amptk. All primary data and data analysis done in this manuscript are available via the Open Science Framework (https://osf.io/4xd9r/). The SynMock sequences and the script to produce them are available in the OSF repository ((https://osf.io/4xd9r/) as well as packaged into AMPtk distributions.

bioinformatics↗

bioRxiv2017

De novo assembly and phasing of dikaryotic genomes from two isolates of Puccinia coronata f. sp. avenae, the causal agent of oat crown rust

Oat crown rust, caused by the fungus Puccinia coronata f. sp. avenae (Pca), is a devastating disease that impacts worldwide oat production. For much of its life cycle, Pca is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two Pca isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for a total assembly length of 99.16 Mbp for 12SD80 and 777 primary contigs with a total length of 105.25 Mbp for 12NC29, and approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of co-expressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in Pca.\n\nImportanceDisease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae (Pca), which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding Pca, resources to study the molecular mechanisms underpinning pathogenicity and emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of Pca as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of Pca.

genomics↗