SYSTEMS AND METHODS FOR IDENTIFICATION OF NUCLEIC ACIDS IN A SAMPLE

• US 20200131506A1
• Filed: 06/21/2018
• Published: 04/30/2020
• Est. Priority Date: 06/21/2017
• Status: Active Application

• Current Assignee: Bluedot, LLC, TRIAD National Security, LLC
• Original Assignee: Bluedot, LLC, TRIAD National Security, LLC
• Inventors: VUYISICH, Momchilo, GANS, Jason, CHAIN, Patrick, HATCH, Andrew
• Application Number:
  US16/621,998
• Publication Number:
  US 20200131506A1
• Time in Patent Office: Days
• 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/40 Population genetics; Linkage disequilibrium
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 35/10 Design of libraries

Abstract

Provided herein are compositions and methods for analyzing nucleic acids in a sample. Compositions include simple barcode sets having reduced DNA sequencing instrument-specific error rates. Methods include methods to deconvolute sequence reads from different samples.

Claims

1. A method of analyzing nucleic acids in a plurality of samples, the method comprising:

purifying nucleic acids from a plurality of samples;

attaching instrument-specific barcode oligonucleotides to the nucleic acids to form a barcoded library of nucleic acid, wherein nucleic acids in a sample receive instrument-specific barcode oligonucleotides sharing a common sequence and wherein different samples receive instrument-specific barcode oligonucleotide with different sequences;

sequencing the barcoded library of nucleic acids with the instrument to obtain sequence data; and

analyzing the sequence data and assigning sequences to samples according to the common instrument-specific barcode sequences.

2. The method of claim 1, wherein analyzing the sequence data further comprises using at least one algorithm or database for microorganisms genomic classification to identify and/or quantify and/or characterize microbes in the sample.

3. The method of claim 1 comprising:

purifying nucleic acids from a sample;

removing non-informative RNA (RNR) from the purified nucleic acids using a sample-specific set of oligonucleotides to form RNR-processed nucleic acids;

preparing a library of nucleic acid fragments from the RNR-processed nucleic acids;

attaching sequence instrument-specific barcode oligonucleotides to the nucleic acid fragments in the library to form a barcoded library of nucleic acid fragments;

sequencing the barcoded library of nucleic acid fragments to obtain sequence data; and

analyzing the sequence data using at least one algorithm or database for microorganisms genomic classification to identify and/or quantify and/or characterize microbes in the sample.

4. A method for generating a plurality of barcodes comprising:

generating, by a processor, a set of possible sequences of length N;

selecting a first barcode sequence from the set of possible sequences;

adding the first barcode sequence to a barcode candidate pool;

identifying a second barcode sequence in the set of possible sequences, wherein, upon sequencing a nucleic acid molecule comprising the second sequence, the second sequence has a reduced likelihood of being incorrectly identified as a different barcode in the barcode candidate pool compared to a threshold value;

adding the second sequence to the barcode candidate pool; and

synthesizing an oligonucleotide library comprising barcodes in the barcode candidate pool.

5. The method of claim 4, further comprising, prior to step (f):

(i) identifying an additional barcode sequence in the set of possible sequences, wherein, upon sequencing a nucleic acid molecule comprising the additional sequence, the additional sequence has a reduced likelihood of being incorrectly identified as a different barcode in the barcode candidate pool compared to the threshold value;

(ii) adding the additional sequence to the barcode candidate pool; and

(iii) repeating steps (i) and (ii) until the barcode candidate pool comprises M sequences, wherein M is at least five, at least 10, at least 20, at least 40, at least 50, at least 75, or at least 100.

6. The method of claim 4, wherein the threshold value is an error tolerance rate, wherein sequence reads of the barcode candidate pool comprising a single error are still correctly identified as a corresponding barcode in the barcode candidate pool.

7. The method of claim 4, wherein (f) is computed in accordance with an error model associated with the sequencer.

8. The method of claim 7, wherein the error model comprises an objective function

9. The method of claim 4 comprising:

(a) generating, by a processor, a set of possible sequences of length N;

(b) selecting a first barcode sequence from the set of possible sequences;

(c) adding the first barcode sequence to a barcode candidate pool;

(d) adding an additional barcode sequence from the set of barcode sequences to the candidate pool, wherein the candidate pool comprising the additional barcode sequence has been identified as having a reduced likelihood that, upon sequencing nucleic acid molecules comprising barcode sequences from the candidate pool, a first member of the barcode candidate pool will be incorrectly identified as a different barcode in the barcode candidate pool compared to the addition of other barcode sequences remaining in the set of possible sequences;

(e) repeating step j until the barcode candidate pool comprises a target number of barcodes M; and

(f) synthesizing an oligonucleotide library comprising barcodes in the barcode candidate pool.

10. The method of claim 9, wherein (d) is computed in accordance with an error model associated with the sequencer.

11. The method of claim 10, wherein the error model associated with the sequencer is determined by sequencing a plurality of training barcode sequences of length N using the sequencer to generate a plurality of sequenced reads;

and

for each position in the training barcode sequences of length N,

counting the number of times each nucleic acid base (A, T, G, C) is observed at a particular position in the plurality of sequence reads; and

generating an error model associated with the sequencer by calculating the probability that a nucleotide observed at a position in a sequence read is accurate.

12. The method of claim 11, wherein the error model comprises an objective function

13. The method of claim 9, wherein

a first barcode of the barcode candidate pool will not be incorrectly identified as a different barcode in the barcode candidate pool when a sequence read of the first barcode differs from a corresponding barcode sequence from the candidate pool by one nucleotide.

18. A group of oligonucleotides made by the method of claim 9.

19. The group of oligonucleotides of claim 18, each oligonucleotide fused to a nucleic acid in a sample by a phosphodiester bond.

21. A method of sequencing a plurality of nucleic acid samples comprising:

attaching oligonucleotides made by the method of claim 9 to nucleic acids in the plurality of nucleic acid samples, wherein the oligonucleotides added to a sample comprise a same sequence and oligonucleotides added to different samples comprise different sequences;

sequencing the plurality of nucleic acid samples to produce a plurality of sequence reads; and

assigning the sequence reads to a sample corresponding to the attached barcode, wherein a sequence read comprising a single error is still correctly assigned to the sample corresponding to the attached barcode.

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

20. (canceled)

22. (canceled)

23. The method of claim 22, wherein the set comprises at least 4^N-15, 4^N-14, 4^N-13, 4^N-12, 4^N-11, 4^N-10, 4^N-9, 4^N-8, 4^N-7, 4^N-6, 4^N-5, 4^N-3, 4^N-2, 4^N-1, or 4^N sequences.

24. The method of claim 22, wherein the barcode sequences are customized to a sequencer, and

wherein the objective function is computed in accordance with an error model associated with the sequencer.

25. The method of claim 24, wherein the error model is computed by:

sequencing a plurality of training barcode sequences of length N using the sequencer to generate a plurality of sequenced reads; and

for each position in the training barcode sequences of length N and for each actual nucleotide in the training barcode sequences, counting the number of occurrences of each nucleotide in the sequenced reads.

26. The method of claim 25, wherein the objective function is:

27. (canceled)

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29. (canceled)

30. A method comprising:

a) generating an initial set of possible nucleotide sequences of length N (4N sequences);

b) in a first iteration, performing a selection process comprising;

i) partitioning the initial set into a first subset comprising one or more sequences selected from the initial set and a second subset comprising remainder sequences of the initial set;

ii) generating pairs of candidate pools and remainder pools, wherein;

1) each candidate pool comprises polynucleotide(s) having the sequence(s) in the first subset and a polynucleotide having the sequence of a different member of the second subset,

2) each remainder pool comprises polynucleotides having sequences of the second subset less the different member added to the candidate pool;

iii) synthesizing oligonucleotides comprising the sequences in the candidate pools;

iv) sequencing the oligonucleotides comprising the sequences in the candidate pools;

v) determining, for each sequenced candidate pool, a sequencing error rate; and

vi) selecting a sequenced candidate pool having an acceptably low error rate or the lowest error rate.

31. The method of claim 30, further comprising

c) in X subsequent iterations, wherein X<

=N−

1, performing a selection process comprising;

i) providing the selected sequenced candidate pool and its paired remainder pool from the previous iteration as a first subset and a second subset, respectively;

ii) generating pairs of candidate pools and remainder pools, wherein;

1) each candidate pool comprises the sequences in the first subset and a different member of the second subset,

2) each remainder pool comprises sequences of the second subset less the different member added to the candidate pool;

iii) synthesizing oligonucleotides comprising the sequences in the candidate pools;

iv) sequencing the oligonucleotides comprising the sequences in the candidate pools;

v) determining, for each sequenced candidate pool, a sequencing error rate; and

vi) selecting a sequenced candidate pool having an acceptably low error rate or the lowest error rate.

32. (canceled)

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38. A method of reducing cross-contamination in a collection comprising a collection of oligonucleotide samples, the method comprising:

a) providing a set of different nucleotide sequences;

b) performing a plurality of oligonucleotide synthesis reactions, each reaction in the plurality producing a synthesis product in which oligonucleotides having a selected nucleotide sequence from the set of nucleotide sequences comprise a predominant oligonucleotide species, wherein;

(1) different synthesis reactions are performed in parallel, wherein reactants and products in each different synthesis reaction travel along different flow paths, or

(2) different synthesis reactions are performed in series, wherein reactants and products in a synthesis reaction follow a flow path and wherein, for each synthesis reaction in the series, conduits in the flow path are replaced or cleaned with a nucleic acid destroying agent; and

c) assembling a collection comprising a plurality of samples, each sample containing a different synthesis product.

39. (canceled)

40. A method of determining cross-contamination of barcode oligonucleotides between different oligonucleotide samples, comprising:

a) providing a plurality of control samples, each control sample comprising one or more predominant control polynucleotide species and having a polynucleotide contamination rate between samples of less than one part per 10,000, one part per 100,000, or one part per 1 million (e.g., prepared by the method of claim 39);

b) providing a collection of oligonucleotide barcode samples, each sample comprising one or more predominant barcode oligonucleotides and contaminating barcode oligonucleotides, wherein the one or more predominant barcodes define a barcode tag;

c) pairing each barcode sample with a different control sample, and attaching the barcode oligonucleotides to control polynucleotides in each pairing to produce sets of barcoded control polynucleotides;

d) pooling the sets of barcoded control polynucleotides;

e) sequencing the barcoded control polynucleotides in the pool to produce sequence information;

f) using the sequence information, quantifying combinations of each barcode tag with each control polynucleotide in a barcoded control polynucleotide, wherein quantities of barcodes mismatched with control polynucleotides indicates cross-contamination.

41. (canceled)

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