BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads

Abstract

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases.

Comprehensive Sequence-Flux Mapping of a Levoglucosan Utilization Pathway in E. coli

“…Longer, more complicated pathways await development of sequencing technologies able to map long DNA stretches with unprecedented accuracy. 47,48 We anticipate such improvements in deep sequencing capability, allowing the use of FluxScan for resolving haplotypes of coupled mutations spread over complete synthetic operons. Reagents, plasmid construction and verification, comprehensive single-site mutagenesis library preparation, growth selections, deep sequencing analysis, biochemical characterization, growth rate and lysate flux measurements of clonal variants, crystallization and structure determination, computational design, accession codes, determination of transport limitations, error approximation for the fitness metric, full DNA and nucleic acid sequences for designs, python scripts, full heatmaps for both selections, fitness metric reproducibility of second selection, distribution of mutations in LGK.9, unselected library frequency distributions, measured versus theoretical flux determination, read coverage statistics, point mutant biochemical properties, specific growth rates of cultures expressing LGK versus LGK.1, fraction ASA buried of improved mutations, LGK design mutations, backcross activities of designs, gene tile PCR primers, and crystallographic statistics.…”

Clinical and biological insights from viral genome sequencing

“…Although there are computational tools 124 to help resolve these issues, new technologies can generate longer reads. Newer, single-molecule sequencers, such as PacBio (Pacific Biosciences) and MinION (Oxford Nanopore), are capable of extremely long-read sequencing, and whole viral genomes (for example, viruses that have genomes less than 20 kb in size, such as Ebola virus, norovirus and influenza A virus) could theoretically be obtained from and selective depletion of DNA with a certain methylation pattern), no similar methods exist, so far, for viral sequencing.…”

Children with cystic fibrosis are infected with multiple subpopulations of Mycobacterium abscessus with different antimicrobial resistance profiles

“…In general, the inference of haplotype frequencies using variant frequencies from short sequencing reads for a microbial population undergoing recombination is a complex problem 15 . However, as we are not attempting to comment on abundances of subpopulations but only their presence, we did not need to infer haplotype frequencies.…”