Methods of sequencing nucleic acids in mixtures and compositions related thereto
View Patent ↗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
1. 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 comprise a palindromic sequence configured to self-hybridize into a double stranded segment, and wherein the double stranded segment comprises a restriction site, wherein the tagging polynucleotides individually comprise an invariant sequence and a random sequence, and wherein the mixing is done under conditions such that the tagging polynucleotides bind the nucleic acids to form nucleic acids individually tagged with the random sequence;
b) circularizing the nucleic acids individually tagged;
c) amplifying with Rolling Circle Amplification (RCA) the nucleic acids individually tagged with random sequences into a mixture of homo-concatemers, wherein the homo-concatemers comprise a repeat of the nucleic acid from the sample and a repeat of the tagging polynucleotide;
d) fragmenting the homo-concatemers to form homo-concatemer fragments
e) cleaving the homo-concatemer fragments with a restriction nuclease to produce cleaved homo-concatemer fragments
f) sequencing the homo-concatemer fragments.
2. The method of claim 1 , wherein the restriction nuclease cleaves a site within the invariant sequence on the tagging polynucleotides.
3. The method of claim 1 , further comprising the step of identifying tagged sequences within the homo-concatemer fragments, separating identical sequences within the random sequence, and reconstructing a nucleic acid sequence that was in the sample.
4. The method of claim 1 ,
wherein the random sequence is within the double stranded segment, and
wherein step e) comprises:
i) mixing the homo-concatemer fragments with a restriction nuclease that cleaves a site correlated to the invariant sequences on the tagging polynucleotides providing cleaved homo-concatemer fragments.
5. A method comprising:
a) providing double stranded nucleic acid fragments comprising a tagging part and a target part, wherein the tagging part comprises invariant sequence and a random sequence, wherein the invariant sequence comprises a first primer site and a restriction site, and wherein the tagging part comprises a palindromic sequence configured to self-hybridize into a double stranded segment, and wherein the double stranded segment comprises a restriction site;
b) mixing the double stranded nucleic acid fragments with a restriction enzyme to the restriction site thereby producing cleaved fragments;
c) mixing the cleaved fragments with single strand RNA/DNA ligase under conditions such that the cleaved fragments form circular fragments;
d) fragmenting 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 thereby producing adaptor nucleic acid conjugates;
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 thereby producing a capture target tagged conjugate; and
g) sequencing the capture target tag conjugate.
6. The method of claim 5 , wherein the random sequence is between the first primer site and the target part.
7. The method of claim 5 , wherein the first primer site is between the random sequence and the target part.
8. The method of claim 5 , wherein the restriction site is between the random sequence and the first primer site.
9. The method of claim 5 , wherein the random sequence is between the restriction site and the first primer site.
10. The method of claim 5 , wherein the nucleic acid fragments comprises two segments of the random sequence wherein the two segments of the random sequence are identical sequences and the restriction site is between the two segments of the random sequence.