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Methods for high-resolution microbiome analysis

A microbial and biological technology, applied in the fields of genomics and metagenomics, which can solve problems such as insufficient discrimination ability and inability to distinguish closely related species and strains with high sequence similarity

Pending Publication Date: 2020-04-21
MT SINAI SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, significant challenges arise in the analysis of metagenomic sequences, often due to the presence of highly similar bacterial strains that differ in relative abundance
Although many metagenomic binning methods have been developed that exploit features that capture sequence composition, organism abundance, and chromosomal organization, many applications still suffer from insufficient discriminative power to distinguish between closely related species with high sequence similarity and strain trouble

Method used

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  • Methods for high-resolution microbiome analysis
  • Methods for high-resolution microbiome analysis

Examples

Experimental program
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Effect test

Embodiment 1

[0248] Example 1: Integrating methylation and composition to bin contigs by lineage

[0249] Epigenetic information was used to isolate contigs assembled from highly similar lines that were indistinguishable using k-mer frequency-based methods. examined the gut microbiota of two groups of children obtained from stool samples from children selected for sequencing based on their high genetic risk of developing T1D.

[0250] Interestingly, it has been observed that the relative abundance of a specific species of Bacteroides that typically dominates the composition of the two samples (i.e., Bacteroides dorsii) is often elevated prior to the onset of T1D in children, making it an Important species for understanding and monitoring. 16S sequencing showed that both samples contained two different strains of Bacteroides multellii: sample A consisted of 63.7% B. dorei) strain 439 (CP008741). Despite the high sequence similarity between the two Bacteroidetes strains (Table 18), each s...

Embodiment 2

[0258] Example 2: Methylome analysis of highly pathogenic Klebsiella pneumoniae strains

[0259] To assess methylome diversity among strains of clinically relevant bacterial species, 878 bacterial lines in the BASET database with methylation motifs identified by SMRT sequencing were analyzed. These included a highly pathogenic and antibiotic-resistant Klebsiella pneumoniae strain (strain 234-12) isolated from patients during the 2011 outbreak in Germany. A single 362 kb plasmid (pKpn23412-362) carried by this line contains 13 genes for antibiotic resistance, including the blaCTX-M-15 (Kpn23412_5431) gene responsible for conferring the extended-spectrum beta-lactamase (ESBL) phenotype on bacteria. Plasmids also contain multiple replicons, which help to expand the range of organisms in which plasmids can successfully replicate.

[0260] The sequence composition profiles of this plasmid and the K. pneumoniae chromosome differ to an extent (Euclidean distance, d = 10.6), which wo...

Embodiment 3

[0261] Example 3: Culture conditions and purification of bacteria from a mixture of eight species

[0262] Bacteroides caccae ATCC 43185, Bacteroides ovatus ATCC 8483, Bacteroides thetaiotaomicron VPI-5482, Bacteroides vulgatus ATCC 8492, Collinsella aerofaciens ) ATCC25986, Clostridium boltae ATCC BAA-613 and Ruminococcus gnavus ATCC 29149 were each cultured in 10 ml of supplemented brain heart infusion in an anaerobic chamber from Coy Laboratory Products. Escherichia coli (Escherichia coli) MG1655 was cultured aerobically in 5ml LB broth. A 10 kb DNA library for SMRT sequencing was constructed according to the manufacturer's instructions.

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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 methvlation signatures inferred from these readsto resolve individual reads and assembled contigs into species- and strain-level clusters. Methods for deconvolving 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.

Description

[0001] Cross References to Related Applications [0002] This patent application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 525,908, filed June 28, 2017, the entire contents of which are incorporated herein by reference. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with the aid of Government support GM114472 awarded by the National Institute of Health. The government has certain rights in this invention. [0005] sequence listing [0006] This application contains a Sequence Listing, which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 19, 2018, is named 242096_000034_SL.txt and is 17,725 bytes in size. technical field [0007] The present subject matter relates generally to the fields of genomics and metagenomics, and in particular to metagenomic binning using DNA methylation and single-molecule long...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B30/10G16B20/00
CPCC12Q1/689C12Q2600/154G16B10/00G16B40/00G16B20/20G16B30/20G16B30/10C12Q1/6869C12N15/1093C12Q2537/164
Inventor G·方J·博劳里埃
Owner MT SINAI SCHOOL OF MEDICINE
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