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US 12,763,164 B2Granted
Methods for high-resolution microbiome analysis
AssigneeIcahn School of Medicine at Mount Sinai
InventorsGang Fang, John Beaulaurier
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Abstract
Methods are presented for binning metagenomic sequences that leverage long reads from a single-molecule long-read sequencing technology and utilize DNA methylation signatures inferred from these reads to resolve individual reads and assembled contigs into species- and strain-level clusters. Methods for deconvoluting prokaryotic organisms in a microbiome sample are presented. Methods for mapping mobile genetic elements to their host organisms in a microbiome sample are also presented.
Claims15
A method of deconvoluting genomes of a plurality of unknown prokaryotic organisms in a microbiome sample, said method comprising the steps of:
- a) sequencing nucleic acids of the plurality of unknown prokaryotic organisms in the microbiome sample obtained from a subject using single-molecule long reads sequencing technology, wherein two or more of the prokaryotic organisms in the microbiome sample have an average nucleotide identity of greater than about 95%, and wherein the sequencing comprises the step of identifying methylated nucleotides, and at least one of the steps of:
- i) sequencing single molecule reads of the nucleic acids;
- and
- ii) assembling contigs from single molecule reads of the nucleic acids;
- b) assigning a methylation score predicting the likelihood and the extent of methylation of the identified methylated nucleotides for sequence motifs of the nucleic acids based on the assembled contigs of the nucleic acids and/or the single molecule reads of the nucleic acids, wherein a methylation score is assigned to the sequence motifs, the sequence motifs comprising at least:
- (i) all possible 4mer, 5mer, and 6mer contiguous sequence motifs, and (ii) all bipartite sequence motifs having 5 or 6 non-specific bases bookended by 3 or 4 specific bases;
- c) removing sequence motifs via motif filtering that do not return a methylation score above a chosen threshold in at least one contig of the assembled contigs of the nucleic acids or on at least twenty unassigned reads for read-level binning, wherein the motif filtering does not identify the single most parsimonious version of any sequence motif;
- d) determining nucleic acid methylation profiles of the assembled contigs of the nucleic acids or the single molecule reads of the nucleic acids in the microbiome sample based on the sequence motifs identified in step (c);
- e) separating the assembled contigs of the nucleic acids and/or the single molecule reads of the nucleic acids into bins corresponding to distinct prokaryotic organisms based on the methylation profiles of step (d);
- and
- f) assembling the bins of step (e), thereby obtaining assembled genomes of the distinct bacterial organisms in the microbiome sample,
- thereby deconvoluting genomes of the prokaryotic organisms in a microbiome sample.
9 more elements
A method of mapping a mobile genetic element to a prokaryotic host organism in a microbiome sample comprising a plurality of unknown prokaryotic organisms, said method comprising the steps of:
- a) sequencing nucleic acids of the plurality of unknown prokaryotic organisms in the microbiome sample obtained from a subject using single-molecule long reads sequencing technology, wherein two or more of the prokaryotic organisms in the microbiome sample have an average nucleotide identity of greater than about 95%, and wherein the sequencing comprises the step of identifying methylated nucleotides and at least one of the steps of
- i) sequencing single molecule reads of the nucleic acids;
- and
- ii) assembling contigs from single molecule reads of the nucleic acids;
- b) assigning a methylation score predicting the likelihood and the extent of methylation of the identified methylated nucleotides for sequence motifs of the nucleic acids on the assembled contigs of the nucleic acids and/or the single molecule reads of the nucleic acids, wherein a methylation score is assigned to the sequence motifs, the sequence motifs comprising at least:
- (i) all possible 4mer, 5mer, and 6mer contiguous sequence motifs, and (ii) all bipartite sequence motifs having 5 or 6 non-specific bases bookended by 3 or 4 specific bases;
- c) removing sequence motifs via motif filtering that do not return a methylation score above a chosen threshold in at least one contig of the assembled contigs of the nucleic acids or on at least twenty unassigned reads for read-level binning, wherein the motif filtering does not identify the single most parsimonious version of any sequence motif;
- d) determining nucleic acid methylation profiles of the assembled contigs of the nucleic acids or the single molecule reads of the nucleic acids of at least one prokaryotic host organism and at least one mobile genetic element in the microbiome sample based on the sequence motifs identified in step (c);
- e) comparing the nucleic acid methylation profiles of the at least one prokaryotic host organism in the microbiome sample and the at least one mobile genetic element in the microbiome sample and determining whether a match exists between said methylation profiles;
- and
- f) repeating steps (d) and (e) until a match between the mobile genetic element and the prokaryotic host organism is identified;
- thereby mapping the mobile genetic element to the prokaryotic host organism.
9 more elements
Dependencies are inferred from the claim text.