Effective machine-learning assembly for next-generation amplicon sequencing with very low coverage

Abstract

BackgroundIn short-read DNA sequencing experiments, the read coverage is a key parameter to successfully assemble the reads and reconstruct the sequence of the input DNA. When coverage is very low, the original sequence reconstruction from the reads can be difficult because of the occurrence of uncovered gaps. Reference guided assembly can then improve these assemblies. However, when the available reference is phylogenetically distant from the sequencing reads, the mapping rate of the reads can be extremely low. Some recent improvements in read mapping approaches aim at modifying the reference according to the reads dynamically. Such approaches can significantly improve the alignment rate of the reads onto distant references but the processing of insertions and deletions remains challenging.ResultsHere, we introduce a new algorithm to update the reference sequence according to previously aligned reads. Substitutions, insertions and deletions are performed in the reference sequence dynamically. We evaluate this approach to assemble a western-grey kangaroo mitochondrial amplicon. Our results show that more reads can be aligned and that this method produces assemblies of length comparable to the truth while limiting error rate when classic approaches fail to recover the correct length. Finally, we discuss how the core algorithm of this method could be improved and combined with other approaches to analyse larger genomic sequences.ConclusionsWe introduced an algorithm to perform dynamic alignment of reads on a distant reference. We showed that such approach can improve the reconstruction of an amplicon compared to classically used bioinformatic pipelines. Although not portable to genomic scale in the current form, we suggested several improvements to be investigated to make this method more flexible and allow dynamic alignment to be used for large genome assemblies.

pgHMA: Application of the heteroduplex mobility assay analysis in phylogenetics and population genetics

“…robustus (Janke et al, 1997), M . fuliginosus (Ranjard et al, 2019), and M . giganteus (Dodt et al, 2016) (GenBank Accession no.…”

“…We conducted 10,000 nonparametric bootstrap (Felsenstein, 1985) (resampled) replicates using NJ method. We also perform phylogenetic analysis on the sequences of mitochondrial control region of these 16 kangaroos with five additional kangaroo species from GenBank, including M. rufus (Nilsson et al, 2018), M. Bernardus (Nilsson et al, 2018), M. robustus (Janke et al, 1997), M. fuliginosus (Ranjard et al, 2019), and M. giganteus (Dodt et al, 2016) (GenBank Accession no. : KY996501, KY996505, Y10524, NC_039717 and NC_027424, respectively).…”