ViPRA-Haplo: De Novo Reconstruction of Viral Populations Using Paired End Sequencing Data.

We present ViPRA-Haplo, a de novo strain-specific assembly workflow for reconstructing viral haplotypes in a viral population from paired-end next generation sequencing (NGS) data. The proposed Viral Path Reconstruction Algorithm (ViPRA) generates a subset of paths from a De Bruijn graph of reads using the pairing information of reads. The paths generated by ViPRA are an over-estimation of the true contigs. We propose two refinement methods to obtain an optimal set of contigs representing viral haplotypes. The first method clusters paths reconstructed by ViPRA using VSEARCH Deorowicz et al. 2015 based on sequence similarity, while the second method, MLEHaplo, generates a maximum likelihood estimate of viral populations. We evaluated our pipeline on both simulated and real viral quasispecies data from HIV (and real data from SARS-COV-2). Experimental results show that ViPRA-Haplo, although still an overestimation in the number of true contigs, outperforms the existing tool, PEHaplo, providing up to 9% better genome coverage on HIV real data. In addition, ViPRA-Haplo also retains higher diversity of the viral population as demonstrated by the presence of a higher percentage of contigs less than 1000 base pairs (bps), which also contain k-mers with counts less than 100 (representing rarer sequences), which are absent in PEHaplo. For SARS-CoV-2 sequencing data, ViPRA-Haplo reconstructs contigs that cover more than 90% of the reference genome and were able to validate known SARS-CoV-2 strains in the sequencing data.

A random forest classifier for detecting rare variants in NGS data from viral populations.

We propose a random forest classifier for detecting rare variants from sequencing errors in Next Generation Sequencing (NGS) data from viral populations. The method utilizes counts of varying length of k-mers from the reads of a viral population to train a Random forest classifier, called MultiRes, that classifies k-mers as erroneous or rare variants. Our algorithm is rooted in concepts from signal processing and uses a frame-based representation of k-mers. Frames are sets of non-orthogonal basis functions that were traditionally used in signal processing for noise removal. We define discrete spatial signals for genomes and sequenced reads, and show that k-mers of a given size constitute a frame. We evaluate MultiRes on simulated and real viral population datasets, which consist of many low frequency variants, and compare it to the error detection methods used in correction tools known in the literature. MultiRes has 4 to 500 times less false positives k-mer predictions compared to other methods, essential for accurate estimation of viral population diversity and their de-novo assembly. It has high recall of the true k-mers, comparable to other error correction methods. MultiRes also has greater than 95% recall for detecting single nucleotide polymorphisms (SNPs) and fewer false positive SNPs, while detecting higher number of rare variants compared to other variant calling methods for viral populations. The software is available freely from the GitHub link https://github.com/raunaq-m/MultiRes.

A Generalized Lattice based Probabilistic Approach for Metagenomic Clustering.

Metagenomics involves the analysis of genomes of microorganisms sampled directly from their environment. Next Generation Sequencing allows a high-throughput sampling of small segments from genomes in the metagenome to generate reads. To study the properties and relationships of the microorganisms present, clustering can be performed based on the inherent composition of the sampled reads for unknown species. We propose a two-dimensional lattice based probabilistic model for clustering metagenomic datasets. The occurrence of a species in the metagenome is estimated using a lattice of probabilistic distributions over small sized genomic sequences. The two dimensions denote distributions for different sizes and groups of words respectively. The lattice structure allows for additional support for a node from its neighbors when the probabilistic support for the species using the parameters of the current node is deemed insufficient. We also show convergence for our algorithm. We test our algorithm on simulated metagenomic data containing bacterial species and observe more than 85% precision. We also evaluate our algorithm on an in vitro-simulated bacterial metagenome and on human patient data, and show a better clustering than other algorithms even for short reads and varied abundance. The software and datasets can be downloaded from https:// github.com/lattclus/lattice-metage.