Search

EP-4736168-A2 - METAGENOMIC DIAGNOSTIC SYSTEMS AND METHODS THEREOF

EP4736168A2EP 4736168 A2EP4736168 A2EP 4736168A2EP-4736168-A2

Abstract

Systems and methods for SwabSeq metagenomic diagnostic platforms are described. The metagenomic diagnostic platforms are meta-genomics based untargeted next generation sequencing (NGS) diagnostic systems for detecting nucleic acids from pathogens. Samples undergo nucleic acid extraction followed by NGS library preparation and sequencing. Results of sequencing can be processed by bioinformatics pipelines. Automation is used to increase accuracy and analyze up to thousands of samples simultaneously without contamination.

Inventors

  • ESKIN, ELEAZAR
  • ARBOLEDA, Valerie

Assignees

  • THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date
20260506
Application Date
20240701

Claims (20)

  1. 1 . A method of detecting a pathogen comprising, obtaining a plurality of nucleic acids from a sample; sequencing the plurality of nucleic acids to obtain a plurality of reads; analyzing the plurality of reads using a bioinformatic process; and identifying the pathogen from the analyzed reads.
  2. 2. The method of claim 1 , further comprising obtaining the sample using at least one of: a swab, a Q-tip, a wipe, and a cloth.
  3. 3. The method of claim 1 , wherein the sample is a tissue, an organ, a bodily fluid, an object, a surface, or a container.
  4. 4. The method of claim 1 , wherein the sample is at least one of: a turbinate swab, a nasal pharyngeal swab, and saliva.
  5. 5. The method of claim 1 , wherein the sample is a clinical sample; wherein the clinical sample is at least one of: urine, a tissue, a skin swab, bronchoalveolar lavage (BAL), sputum, and blood.
  6. 6. The method of claim 1 , wherein the sample is an upper respiratory specimen or a lower respiratory specimen from a subject.
  7. 7. The method of claim 1 , wherein the obtaining step obtains the plurality of nucleic acids using at least one nucleic acid extraction kit.
  8. 8. The method of claim 1 , further comprising adding a known quantity of Bacteriophage MS2 to the sample.
  9. 9. The method of claim 1 , further comprising constructing at least one next generation sequencing library.
  10. 10. The method of claim 9, wherein the at least one next generation sequencing library is at least one of: a DNA library, and an RNA library.
  11. 11 . The method of claim 9, wherein the sequencing step uses the at least one next generation sequencing library.
  12. 12. The method of claim 1 , wherein the analyzing step comprises matching a plurality of sequencing reads to at least one pathogen sequence in a database.
  13. 13. The method of claim 12, further comprising generating a plurality of simulated reads using a set of curated pathogen sequences; analyzing the plurality of simulated reads using the database to check if the database contains an error; and eliminating a plurality of false reads in the database by analyzing the plurality of simulated reads.
  14. 14. The method of claim 8, further comprising using the plurality of reads of the pathogen and a plurality of reads of a control added prior to nucleic acid extraction or a plurality of reads of a control added after nucleic acid extraction to quantify an amount of the sample.
  15. 15. The method of claim 14, wherein the control is Bacteriophage MS2.
  16. 16. The method of claim 1 , wherein the pathogen is a pathogenic organism, a bacterium, a virus, or a fungus.
  17. 17. The method of claim 1 further comprising processing a plurality of samples at a time using automation.
  18. 18. The method of claim 1 , further comprising processing a plurality of samples at a time using automation by interleaving a plurality sets of samples to increase a number that is processed on an automated instrument.
  19. 19. The method of claim 1 , further comprising processing a plurality of samples using at least one microfluidic liquid handler.
  20. 20. The method of claim 18, wherein automation eliminates cross-sample contamination when processing the plurality of samples.

Description

Metagenomic Diagnostic Systems and Methods Thereof CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The current application claims the benefit of and priority under 35 U.S.C. § 119 (e) to U.S. Provisional Patent Application No. 63/511 ,532 entitled “Metagenomic Diagnostic Systems and Methods Thereof” filed June 30, 2023. The disclosure of U.S. Provisional Patent Application No. 63/511 ,532 is hereby incorporated by reference in its entirety for all purposes. STATEMENT OF FEDERAL FUNDING [0002] This invention was made with government support under 75A50122C00033 awarded by the U.S. Department of Health and Human Services. The government has certain rights in the invention. SEQUENCE LISTING [0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 1 , 2024, is named R4-08645.PCT.xml and is 3 kilobytes in size. FIELD OF THE INVENTION [0004] The present invention generally relates to systems and methods for metagenomic diagnosis. BACKGROUND [0005] Culturing is a conventional method to identify pathogens. However, culturing is time-consuming and many pathogens that need specific culture conditions are difficult to grow. Metagenomic sequencing is a target-independent approach that offers a comprehensive view of the pathogenic agents and provides a detection of common and unexpected pathogens in samples. This technology performs well in detecting rare pathogens and/or novel viral strains. Current approaches to metagenomic sequencing can only process a small number of samples at a time and each sample costs hundreds of dollars. There are multiple challenges to processing larger amounts of samples including difficulty to prepare the samples and cross-contamination which is when the samples are mixed during processing causing incorrect results. BRIEF SUMMARY [0006] Many embodiments are directed to systems and methods for metagenomic diagnosis. [0007] Some embodiments include a method of detecting a pathogen comprising, obtaining a plurality of nucleic acids from a sample; sequencing the plurality of nucleic acids to obtain a plurality of reads; analyzing the plurality of reads using a bioinformatic process; and identifying the pathogen from the analyzed reads. [0008] Some embodiments further comprise obtaining the sample using at least one of: a swab, a Q-tip, a wipe, and a cloth. [0009] In some embodiments, the sample is a tissue, an organ, a bodily fluid, an object, a surface, or a container. [0010] In some embodiments, the sample is at least one of: a turbinate swab, a nasal pharyngeal swab, and saliva. [0011] In some embodiments, the sample is a clinical sample; wherein the clinical sample is at least one of: urine, a tissue, a skin swab, bronchoalveolar lavage (BAL), sputum, and blood. [0012] In some embodiments, the sample is an upper respiratory specimen or a lower respiratory specimen from a subject. [0013] In some embodiments, the obtaining step obtains the plurality of nucleic acids using at least one nucleic acid extraction kit. [0014] Some embodiments further comprise adding a known quantity of Bacteriophage MS2 to the sample. [0015] Some embodiments further comprise constructing at least one next generation sequencing library. [0016] In some embodiments, the at least one next generation sequencing library is at least one of: a DNA library, and an RNA library. [0017] In some embodiments, the sequencing step uses the at least one next generation sequencing library. [0018] In some embodiments, the analyzing step comprises matching a plurality of sequencing reads to at least one pathogen sequence in a database. [0019] Some embodiments further comprise generating a plurality of simulated reads using a set of curated pathogen sequences; analyzing the plurality of simulated reads using the database to check if the database contains an error; and eliminating a plurality of false reads in the database by analyzing the plurality of simulated reads. [0020] Some embodiments further comprise using the plurality of reads of the pathogen and a plurality of reads of a control added prior to nucleic acid extraction or a plurality of reads of a control added after nucleic acid extraction to quantify an amount of the sample. [0021] In some embodiments, the control is Bacteriophage MS2. [0022] In some embodiments, the pathogen is a pathogenic organism, a bacterium, a virus, or a fungus. [0023] Some embodiments further comprise processing a plurality of samples at a time using automation. [0024] Some embodiments further comprise processing a plurality of samples at a time using automation by interleaving a plurality sets of samples to increase a number that is processed on an automated instrument. [0025] Some embodiments further comprise processing a plurality of samples using at least one microfluidic liquid handler. [0026] In some embodiments, automation eliminates cross-sample co