Method and system for fragment assembly and sequence identification

• US 10,255,990 B2
• Filed: 11/13/2017
• Issued: 04/09/2019
• Est. Priority Date: 11/11/2016
• Status: Active Grant

• Current Assignee: Psomagen, Inc.
• Original Assignee: uBiome, Inc.
• Inventors: Apte, Zachary, Richman, Jessica, Almonacid, Daniel, Ortiz, Rodrigo
• Primary Examiner(s): Harward, Soren
• Application Number:
  US15/811,544
• Publication Number:
  US 20180137240A1
• Time in Patent Office: 512 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

Embodiments of a method and/or system for improving fragment assembly and/or sequence identification includes: collecting a sample including a set of nucleic acid components associated with a set of microorganisms; generating a set of tagged sequence fragments; amplifying the set of tagged sequence fragments and sequencing the set of tagged sequence fragments; based upon the set of identifier tags, generating a set of branched assemblies of candidate sequence fragments, wherein each of the set of branched assemblies includes a set of ordered nodes and a set of branches distributed across the set of nodes; implementing a threshold criterion to reduce the set of branched assemblies to a set of branch-reduced assemblies; and identifying a set of sequences corresponding to the set of branch-reduced assemblies and/or generating an analysis informative of the set of microorganisms associated with the sample.

Claims

1. A method for improving fragment assembly and sequence identification in relation to a set of microorganisms, the method comprising:

collecting a sample including a set of nucleic acid components associated with the set of microorganisms;

generating a set of tagged sequence fragments based on a set of identifier tags and a set of sequence fragments derived from the set of nucleic acid components of the sample, wherein generating the set of tagged sequence fragments comprises;

targeting, with guide RNAs (gRNAs) of gRNA complexes, microorganism target sequences of the set of nucleic acid components at identified sequence positions;

cutting the set of nucleic acid components in relation to the microorganism target sequences with endonucleases of the gRNA complexes, thereby generating the set of sequence fragments;

ligating the set of identifier tags to the set of sequence fragments, thereby generating augmented sequence fragments, wherein each identifier tag of the set of identifier tags comprises a complete barcode sequence comprising a first barcode portion and a second barcode portion separable by cutting of a cutting site; and

generating the set of tagged sequence fragments based on circularization of the augmented sequence fragments and the cutting of the cutting sites to separate the first barcode portions and the second barcode portions;

sequencing the set of tagged sequence fragments;

generating a set of branched assemblies corresponding to candidate sequence fragments, based on the identified sequence positions and the set of identifier tags associated with the sequenced set of tagged sequence fragments, wherein each branched assembly of the set of branched assemblies comprises a set of branches distributed across a set of nodes, wherein generating the set of branched assemblies comprises;

detecting the first barcode portions and the second barcode portions of the set of identifier tags at the sequenced set of tagged sequence fragments; and

generating the set of branches distributed across the set of nodes based on the detected first barcode portions and the second barcode portions in relation to the complete barcode sequences;

implementing a threshold criterion to reduce the set of branched assemblies to a set of branch-reduced assemblies; and

identifying a set of microorganism sequences corresponding to the set of branch-reduced assemblies.

2. The method of claim 1, wherein implementing the threshold criterion comprises, for the each branched assembly of the set of branched assemblies, applying the threshold criterion for each node coupled to branches of the set of branches to generate an unbranched assembly.

3. The method of claim 1, wherein each node of the set of nodes of the set of branched assemblies corresponds to a microorganism sequenced fragment comprising a length, an initiating position, and a terminating position, wherein a connection between adjacent nodes of the set of nodes corresponds to a candidate continuity between a plurality of microorganism sequenced fragments, and wherein identifying the set of microorganism sequences comprises identifying the set of microorganism sequences based on the length, the initiating position, the terminating position, and the connection between adjacent nodes.

4. The method of claim 1, wherein each node of the set of nodes corresponds to a position along an assembled microorganism sequence, wherein each branch of the set of branches corresponds to a microorganism sequenced fragment comprising an initial position corresponding to an upstream node and a terminating position corresponding to a downstream node, and wherein identifying the set of microorganism sequences comprises identifying the set of microorganism sequences based on the initial position, the terminating position, and the position along the assembled microorganism sequence.

5. The method of claim 1, wherein the threshold criterion comprises a similarity coefficient associated with similarity between the set of branched assemblies and an additional set of branched assemblies corresponding to an additional sample, and wherein implementing the threshold criterion comprises implementing the similarity coefficient for the set of branches and the set of nodes to reduce the set of branched assemblies to the set of branch-reduced assemblies.

6. The method of claim 5, wherein the similarity coefficient comprises at least one of:

a Tanimoto Coefficient, a Jaccard distance parameter, a Sorensen similarity index, a simple matching coefficient, a Mountford'"'"'s index of similarity, a Hamming distance, a Dice'"'"'s coefficient, a Tversky index, and a Mutual information parameter.

7. The method of claim 1, wherein the set of nucleic acid components comprise target sequence regions associated with the set of microorganisms, wherein the target sequence regions comprise target sequences for identification of a specific target that include at least one of a 16S rRNA sequence region, 18S rRNA sequence region, and an ITS sequence region, and wherein generating the set of branched assemblies comprises generating the set of branched assemblies for facilitating the improved fragment assembly and the sequence identification in relation to the target sequence regions.

8. The method of claim 7, wherein the target sequence regions comprise a polymorphic or variable region of a specific target sequence region, wherein the polymorphic region can be used for identification, selection and/or differentiation of a single microorganisms.

9. The method of claim 8, wherein the specific target sequence region comprises:

16S sequence region, 18S sequence region, ITS region, and any sequence region that can be used for identification, selection and/or differentiation of a single microorganisms.

10. The method of claim 8, wherein the target sequence regions comprise at least one polymorphic region of the 16S rRNA sequence region, wherein the at least one polymorphic region comprises one or more of:

V1, V2, V3, V4, v5, v6, v7, v8, and v9.

11. The method of claim 1, further comprising:

generating a graphical representation of the set of branched assemblies based on the set of branches and the set of nodes;

presenting the graphical representation of the set of branched assemblies;

collecting a user input associated with the set of branched assemblies in response to presenting the graphical representation of the set of branched assemblies, wherein implementing the threshold criterion comprises implementing the threshold criterion to reduce the set of branched assemblies to the set of branch-reduced assemblies based on the user input.

12. The method of claim 1, wherein each branched assembly of the set of branched assemblies corresponds to a set of candidate assembled sequences comprising de-branched assembled sequences, and wherein the method further comprises defining an operational microorganism taxonomic unit comprising the de-branched assembled sequences.

13. The method of claim 1, wherein the endonucleases of the gRNA complexes comprise Cas9 endonucleases.

14. The method of claim 1, wherein the complete barcode sequences comprise unique complete barcode sequences operable to uniquely identify the microorganism target sequences.