Optimal index sequences for multiplex massively parallel sequencing
AssigneeIllumina, Inc.
InventorsRyan Kelley, Gordon Bean, Eric Vermaas
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Abstract
The disclosed embodiments concern index oligonucleotides configured to identify sources of samples of nucleic acids and methods, apparatus, systems and computer program products for identifying and making the index oligonucleotides. In some implementations, the index oligonucleotides include a set of index sequences, a Hamming distance between any two index sequences of the set of index sequences meeting one or more criteria. System, apparatus, and computer program products are also provided for determining a sequence of interest using the index oligonucleotides.
Claims27
A set of oligonucleotides comprising a plurality of subsets of oligonucleotides, wherein:
- the set of oligonucleotides is configured to identify sources of nucleic acid samples in multiplex massively parallel sequencing, each of the nucleic acid samples comprising a plurality of nucleic acid molecules;
- the set of oligonucleotides comprises a set of index sequences comprising at least 6 different index sequences, each subset of the plurality of subsets of oligonucleotides comprising a plurality of index sequences of the set of index sequences;
- a Hamming distance between any two index sequences of the set of index sequences is not less than a first criterion value, wherein the first criterion value is at least 2;
- and
- the set of index sequences comprises a plurality of pairs of color-balanced index sequences, wherein any two bases at corresponding sequence positions of each pair of color-balanced index sequences include both (i) an adenine (A) base or a cytosine (C) base, and (ii) a guanine (G) base, a thymine (T) base, or a uracil (U) base.
A method for making a plurality of oligonucleotides for multiplex massively parallel sequencing, the method comprising:
- (a) selecting a set of index sequences from a pool of different index sequences, wherein
- the set of index sequences comprise at least 6 different sequences;
- a Hamming distance between any two index sequences in the set of index sequences is not less than a first criterion value, wherein the first criterion value is at least 2, and
- the set of index sequences comprises a plurality of pairs of color-balanced index sequences, wherein any two bases at corresponding sequence positions of each pair of color-balanced index sequences include both (i) an adenine base or a cytosine base, and (ii) a guanine base, a thymine base, or a uracil base;
- and
- (b) synthesizing the plurality of oligonucleotides comprising the set of index sequences.
A computer program product comprising a non-transitory machine readable medium storing program code that, when executed by one or more processors of a computer system, causes the computer system to implement a method for selecting a set of index sequences to be incorporated in a set of oligonucleotides configured to be used to in multiplex massively parallel sequencing, said program code comprising:
- (a) code for adding to a candidate set of index sequences a randomly chosen pair of color-balanced index sequences from a pool of different index sequences, wherein any two bases at corresponding sequence positions of each pair of color-balanced index sequences include both (i) an adenine base or a cytosine base, and (ii) a guanine base, a thymine base, or a uracil base;
- (b) code for sorting index sequences remaining in the pool of index sequences based on minimum Hamming distance to members in the candidate set;
- (c) code for removing any remaining index sequence whose minimum Hamming distance to the members in the candidate set is less than a first criterion value or minimum edit distance to the members in the candidate set is less than a second criterion value;
- (d) code for repeating (a)-(c) to maximize a size of the candidate set;
- and
- (e) code for selecting from the candidate set the set of index sequences to be incorporated into the set of oligonucleotides configured to be used in multiplex massively parallel sequencing.
Dependencies are inferred from the claim text.