EP-4524255-B1 - METHODS AND COMPOSITIONS FOR NUCLEIC ACID ANALYSIS

Inventors

• ZHENG, Xinying
• SAXONOV, SERGE
• SCHNALL-LEVIN, MICHAEL
• NESS, KEVIN
• BHARADWAJ, RAJIV

Dates

Publication Date

20260513

Application Date

20161202

Claims (12)

1. A method of analyzing nucleic acids obtained from a FFPE tissue sample while maintaining spatial context, comprising: a) extracting nucleic acids from a plurality of regions of the FFPE tissue sample; b) following (a), partitioning the extracted nucleic acids into a plurality of wells; wherein nucleic acids in spatial proximity to each other in the FFPE tissue sample are introduced into the same well; c) barcoding the partitioned nucleic acids with partition-specific barcode sequences to form a plurality of barcoded nucleic acids; d) obtaining sequence information from the plurality of barcoded nucleic acids, wherein the sequence information from the plurality of barcoded nucleic acids comprises sequence information of the partition-specific barcode sequences; and e) spatially resolving the plurality of barcoded nucleic acids to a region of spatial proximity in the FFPE tissue sample, wherein barcoded nucleic acids derived from a region of spatial proximity in the FFPE tissue sample comprise the same partition-specific barcode sequence.
2. The method of claim 1, wherein the barcoding comprises amplifying with a primer comprising a barcode sequence.
3. The method of claim 1 or 2, wherein at least two of the nucleic acids partitioned into the same well in the partitioning b) step comprise different sequences.
4. The method of any one of claims 1-3, further comprising imaging the FFPE tissue sample.
5. The method of any one of claims 1-4, wherein the FFPE tissue sample is a cancer tissue sample.
6. The method of any one of claims 1-5, wherein the barcoded nucleic acids in different wells are pooled prior to d) obtaining sequence information.
7. The method of any one of claims 1-6, wherein the sequence information further comprises information relating to a nucleic acid obtained from the FFPE tissue sample.
8. The method of any one of claims 1-7, wherein the nucleic acids extracted from the FFPE tissue sample comprise nucleic acid tags previously applied to the sample.
9. The method of any one of claims 1-8, wherein the obtaining sequence information comprises high throughput sequencing of the plurality of barcoded nucleic acids.
10. The method of any one of claims 1-9, wherein a partition-specific barcode sequence in a given well comprises two or more separate subsequences.
11. The method of claim 4, wherein the FFPE tissue sample is imaged with optically identifiable tags.
12. The method of claim 11, wherein e) further comprises correlating sequences of the barcoded nucleic acids derived from a region of spatial proximity with the imaged optically identifiable tags.

Description

BACKGROUND OF THE INVENTION Polynucleotide sequencing continues to find increasing use in medical applications such as genetic screening and genotyping of tumors. Many polynucleotide sequencing methods rely on sample processing techniques of the original sample, including random fragmentation of polynucleotides. These processing techniques can provide advantages in terms of throughput and efficiency, but the resultant sequence information obtained from these processed samples can lack important contextual information in terms of the location of particular sequences within the broader linear (two-dimensional) sequence of the original nucleic acid molecule that contained those sequences. Structural context within the three dimensional space of the original sample is also lost with many sample processing and sequencing techniques. There is thus a need for sequencing technologies that retain structural and molecular context of the identified nucleic acid sequences. EP2881465 relates to a method, a device, and an apparatus for analyzing the expression of a gene in single cells. SUMMARY OF THE INVENTION Accordingly, the present invention provides methods, systems and compositions for providing sequence information that retains both molecular and structural context of the originating nucleic acid molecule. The present disclosure provides methods of analyzing nucleic acids obtained from a FFPE tissue sample while maintaining spatial context. Such methods include the steps of: a) extracting nucleic acids from a plurality of regions of the FFPE tissue sample; b) following (a), partitioning the extracted nucleic acids into a plurality of wells; wherein nucleic acids in spatial proximity to each other in the FFPE tissue sample are introduced into the same well;c) barcoding the partitioned nucleic acids with partition-specific barcode sequences to form a plurality of barcoded nucleic acids;d) obtaining sequence information from the plurality of barcoded nucleic acids, wherein the sequence information from the plurality of barcoded nucleic acids comprises sequence information of the partition-specific barcode sequences; ande) spatially resolving the plurality of barcoded nucleic acids to a region of spatial proximity in the FFPE tissue sample, wherein barcoded nucleic acids derived from a region of spatial proximity in the FFPE tissue sample comprise the same partition-specific barcode sequence. In some embodiments, the barcoding comprises amplifying with a primer comprising a barcode sequence. In some embodiments, at least two of the nucleic acids partitioned into the same well in the partitioning b) step comprise different sequences. In some embodiments, the method further comprising imaging the FFPE tissue sample. In some embodiments the FFPE tissue sample is a cancer tissue sample. In some embodiments, the barcoded nucleic acids in different wells are pooled prior to d) obtaining sequence information. In some embodiments, the sequence information further comprises information relating to a nucleic acid obtained from the FFPE tissue sample. In some embodiments, the nucleic acids extracted from the FFPE tissue sample comprise nucleic acid tags previously applied to the sample. In some embodiments, the obtaining sequence information comprises high throughput sequencing of the plurality of barcoded nucleic acids. In some embodiments, a partition-specific barcode sequence in a given well comprises two or more separate subsequences. In some embodiments, the FFPE tissue sample is imaged with optically identifiable tags. In some embodiments, e) further comprises correlating sequences of the barcoded nucleic acids derived from a region of spatial proximity with the imaged optically identifiable tags. BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 provides a schematic illustration of molecular context and structural context in accordance with the methods described herein.FIGURE 2 provides a schematic illustration of a process described herein.FIGURE 3 illustrates a typical workflow for performing an assay to detect sequence information, using the methods and compositions disclosed herein.FIGURE 4 provides a schematic illustration of a process for combining a nucleic acid sample with beads and partitioning the nucleic acids and beads into discrete droplets.FIGURE 5 provides a schematic illustration of a process for barcoding and amplification of chromosomal nucleic acid fragments.FIGURE 6 provides a schematic illustration of the use of barcoding of nucleic acid fragments in attributing sequence data to their originating source nucleic acid molecule.FIGURE 7 provides a schematic illustration of an exemplary sample preparation method. DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which a