Adaptors for nucleic acid constructs in transmembrane sequencing
The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.
1. A method of obtaining a single molecule consensus sequence and identifying the genomic nucleic acid source of a plurality of template nucleic acid molecules derived from genomic nucleic acids from at least two sources, comprising:
(a) preparing the plurality of template nucleic acid molecules from genomic nucleic acids from at least two sources, comprising:
i) providing double stranded nucleic acid templates from the genomic nucleic acids from at least two sources, wherein each of the double stranded nucleic acid templates comprises a first strand having a 3′ end and a 5′ end and a second strand complementary to the first strand and having a 3′ end and a 5′ end;
ii) ligating a first chemically synthesized DNA hairpin loop to each of the double stranded nucleic acid templates to covalently link the 3′ end of the first strand to the 5′ end of the second strand; and
iii) ligating a second chemically synthesized DNA hairpin loop to each of the double stranded nucleic acid templates to covalently link the 5′ end of the first strand to the 3′ end of the second strand,
wherein each of the template nucleic acid molecules further comprises a region of recognizable artificial nucleic acid sequence between each of the double stranded templates and one of the first chemically synthesized DNA hairpin loop and the second chemically synthesized DNA hairpin loop,
(b) pooling the plurality of template nucleic acid molecules from the at least two sources of step (a) together to produce unamplified pooled template nucleic acid molecules; and
purifying the pooled unamplified template nucleic acid molecules by binding of either the first hairpin loop or the second hairpin loop to a surface;
(c) performing a single molecule sequencing on the pooled unamplified template nucleic acid molecules bound to the surface to produce sequence data;
(d) comparing the sequence data from the first strand and the second strand of the pooled unamplified template nucleic acid molecules, thereby obtaining a single molecule consensus sequence for each of the template nucleic acid molecules: and
(e) performing a multiplex sequence analysis to identify the genomic nucleic acid source of each of the template nucleic acid molecules using the sequence data of the region of recognizable artificial nucleic acid sequence of each of the template nucleic acid molecules.
2. The method of claim 1 , wherein said sequence data comprises a sequence of the first chemically synthesized DNA hairpin loop and the second chemically synthesized DNA hairpin loop.
3. The method of claim 1 , wherein said single-molecule sequencing is performed using a sequencing by synthesis technology.
4. The method of claim 3 , wherein said sequencing by synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process.
5. The method of claim 1 , wherein said single-molecule sequencing is performed using a nanopore sensor.
6. The method of claim 1 , wherein said sequence data comprises the sequence of at least 1000 bases.