Methods of sequencing nucleic acids in mixtures and compositions related thereto

• US 10,227,584 B2
• Filed: 02/17/2014
• Issued: 03/12/2019
• Est. Priority Date: 02/20/2013
• Status: Active Grant

• Current Assignee: Emory University, Johns Hopkins University
• Original Assignee: Emory University, Johns Hopkins University
• Inventors: Emerick, Mark C., Agnew, William S.
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/768,749
• Publication Number:
  US 20160046930A1
• Time in Patent Office: 1,849 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

This disclosure relates to analyzing the end-to-end sequence and the relative distributions in heterogeneous mixtures of polynucleotides and methods and enabling reagents related thereto. In certain embodiments this method relates to the complete full length sequencing and quantitative profiling of mRNAs present in the transcriptomes of cells or tissues of but not limited to, higher multicellular organisms that possess interrupted genes subject to complex post-transcriptional RNA processing.

Claims

1. A method comprising:

a) providing double stranded nucleic acid fragments comprising a tagging part and a target part, wherein the tagging part comprises a segment of overlapping sequences and a segment of varying sequences, wherein the overlapping sequences comprise a first primer site and a restriction site,

b) mixing the double stranded fragments with a restriction enzyme to the restriction site providing cleaved fragments;

c) mixing the cleaved fragments with an enzyme under conditions such that the cleaved fragments form circular fragments;

d) breaking the circular fragments at random points providing sheared fragments;

e) ligating an adaptor to the ends of the double stranded nucleic acids wherein the adaptor comprises a second primer site providing an adaptor nucleic acid conjugate;

f) amplifying the adaptor nucleic acid conjugates with primers to the first and second primer sites, wherein the first primer comprises a first capture sequence on the 5′

end and the second primer comprises a second capture sequence on the 5′

end to provide a capture target tagged conjugate; and

g) sequencing the capture target tag conjugate.

2. The method of claim 1, wherein the segment of varying sequences is between the first primer site and the target part.

3. The method of claim 1, wherein the first primer site is between the segment of varying sequences and the target part.

4. The method of claim 1, wherein the restriction site is between the segment of varying sequences and the first primer site.

5. The method of claim 1, wherein the segment of varying sequences is between the restriction site and the first primer site.

6. The method of claim 1, wherein the nucleic acid fragments comprises two segments of varying sequences wherein the varying segments are identical sequences and the restriction site is between the identical sequences.

7. A method comprising

a) mixing a sample and a group of tagging polynucleotides, wherein the sample comprises a mixture of nucleic acids of different length and/or different sequence, wherein the tagging polynucleotides individually comprise overlapping sequences and a part with random sequences and wherein the tagging polynucleotides comprise a palindromic sequence configured to self-hybridize into a double stranded segment wherein the double stranded segment comprises a restriction site, wherein the part with random sequences is within the double stranded segment, and wherein the mixing is done under conditions such that the tagging polynucleotides bind the nucleic acids to form nucleic acids individually tagged with random sequences;

b) replicating the nucleic acid mixture individually tagged with random sequences into a mixture of homopolymers, wherein the homopolymers comprise a repeating nucleic acid and a repeating sequence tag;

c) mixing the homopolymer fragments with a restriction nuclease that cleaves a site correlated to the overlapping sequences on the tagging polynucleotides providing cleaved homopolymer fragments; and

d) sequencing the cleaved homopolymer fragments.