SAMPLE INDEXING METHODS AND COMPOSITIONS FOR SEQUENCING APPLICATIONS

• US 20160314242A1
• Filed: 04/22/2016
• Published: 10/27/2016
• Est. Priority Date: 04/23/2015
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: SCHNALL-LEVIN, MICHAEL, GREENFIELD, LAWRENCE
• Application Number:
  US15/135,858
• Publication Number:
  US 20160314242A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Compositions, processes and systems are provided for preparing and analyzing sample indexing of nucleic acid libraries for multiplexed sequencing analysis of diverse sample sets.

Claims

1. A universal sample index library, comprising a plurality of sets of sample index oligonucleotides, each of the plurality of sets of sample index oligonucleotides comprises a plurality of individual sample index oligonucleotide sequences, wherein:

the sample index oligonucleotides in each of the plurality of sets of sample index oligonucleotides are different from sample index oligonucleotides in each other set of sample index oligonucleotides; and

each sample index oligonucleotide sequence within a set of sample index oligonucleotides comprises a different nucleotide sequence from each other sample index oligonucleotide in the same set of sample index oligonucleotides.

2. The library of claim 1, wherein each set of sample index oligonucleotides comprises at least three, four, five, six, seven, eight, nine, or ten different sample index oligonucleotides.

3. The library of claim 1, wherein the plurality of sets of sample index oligonucleotides comprises at least about 10 sets, 20 sets, 50 sets, or 100 sets.

4. The library of claim 3, wherein each of the plurality of sets of sample index oligonucleotides has complete diversity from other sets of the plurality.

5. The library of claim 1, wherein each sample index oligonucleotide within a set of sample index oligonucleotides comprises a different nucleotide at each sequence position from each other sample index oligonucleotide in the set of sample index oligonucleotides.

6. The library of claim 1, wherein each sample index oligonucleotide within a set of sample index oligonucleotides does not share a common 4-mer sequence with any other sample index oligonucleotide within that same set of sample index oligonucleotides.

7. The library of claim 1, wherein sample index oligonucleotides within a set have less than 80% common bases at common sequence positions with other sample index oligonucleotides within the same set.

8. The library of claim 1, wherein the sample index oligonucleotides are from about 4 to about 10 bases in length.

9. The library of claim 1, wherein the sample index library further comprises adapter sequences containing additional sequence elements.

10. The library of claim 8, wherein the sample index oligonucleotides are integrated into the adapter sequences.

11. A method of sample indexing oligonucleotides for nucleic acid sequencing, comprising:

providing a plurality of sequencing libraries of oligonucleotides, each of the plurality of sequencing libraries being prepared from a different sample;

attaching sets of sample index oligonucleotides to each of the plurality of sequencing libraries of oligonucleotides, wherein

the sample index oligonucleotides in each of the plurality of sets of sample index oligonucleotides are different from sample index oligonucleotides in each other set of sample index oligonucleotides; and

each sample index oligonucleotide sequence within a set of sample index oligonucleotides comprises a different nucleotide sequence from each other sample index oligonucleotide in the set of sample index oligonucleotides.

12. The method of claim 11, wherein each sample index oligonucleotide sequence within a set of sample index oligonucleotide sequences comprises a different nucleotide at each sequence position from each other sample index oligonucleotide in the set of sample index oligonucleotides.

13. The method of claim 11, wherein the sets of sample index oligonucleotides comprise at least about 10 sets, 20 sets, 50 sets, or 100 sets.

14. The method of claim 11, wherein each set of sample index oligonucleotides has complete diversity from the other sets of sample index oligonucleotides.

15. The method of claim 11, wherein each sample index oligonucleotide within a set of sample index oligonucleotides does not share a common 4-mer sequence with any other sample index oligonucleotide within that same set of sample index oligonucleotides.

16. The method of claim 11, wherein sample index oligonucleotides within a set have less than 80% common bases at common sequence positions with other sample index oligonucleotides within the same set.

17. The method of claim 11, wherein the sample index oligonucleotides are from about 4 to about 10 bases in length.

18. The method of claim 17, wherein sample index oligonucleotides of different sets have different lengths.

19. The method of claim 11, wherein subsequent to the attaching step, the sequencing libraries of oligonucleotides are pooled together and subjected to a sequencing process.

20. The method of claim 11, wherein the sample index oligonucleotide sequences are further integrated into adapter sequences comprising additional sequence elements.