An assembly-free method of phylogeny reconstruction using short-read sequences from pooled samples without barcodes.

A current strategy for obtaining haplotype information from several individuals involves short-read sequencing of pooled amplicons, where fragments from each individual is identified by a unique DNA barcode. In this paper, we report a new method to recover the phylogeny of haplotypes from short-read sequences obtained using pooled amplicons from a mixture of individuals, without barcoding. The method, AFPhyloMix, accepts an alignment of the mixture of reads against a reference sequence, obtains the single-nucleotide-polymorphisms (SNP) patterns along the alignment, and constructs the phylogenetic tree according to the SNP patterns. AFPhyloMix adopts a Bayesian inference model to estimate the phylogeny of the haplotypes and their relative abundances, given that the number of haplotypes is known. In our simulations, AFPhyloMix achieved at least 80% accuracy at recovering the phylogenies and relative abundances of the constituent haplotypes, for mixtures with up to 15 haplotypes. AFPhyloMix also worked well on a real data set of kangaroo mitochondrial DNA sequences.

Reassembling haplotypes in a mixture of pooled amplicons when the relative concentrations are known: A proof-of-concept study on the efficient design of next-generation sequencing strategies.

Next-generation sequencing can be costly and labour intensive. Usually, the sequencing cost per sample is reduced by pooling amplified DNA = amplicons) derived from different individuals on the same sequencing lane. Barcodes unique to each amplicon permit short-read sequences to be assigned appropriately. However, the cost of the library preparation increases with the number of barcodes used. We propose an alternative to barcoding: by using different known proportions of individually-derived amplicons in a pooled sample, each is characterised a priori by an expected depth of coverage. We have developed a Hidden Markov Model that uses these expected proportions to reconstruct the input sequences. We apply this method to pools of mitochondrial DNA amplicons extracted from kangaroo meat, genus Macropus. Our experiments indicate that the sequence coverage can be efficiently used to index the short-reads and that we can reassemble the input haplotypes when secondary factors impacting the coverage are controlled. We therefore demonstrate that, by combining our approach with standard barcoding, the cost of the library preparation is reduced to a third.