Method for generating extended sequence reads
The present invention provides an approach to increase the effective read length of commercially available sequencing platforms to several kilobases and be broadly applied to obtain long sequence reads from mixed template populations. A method for generating extended sequence reads of long DNA molecules in a sample, comprising the steps of assigning a specific barcode sequence to each template DNA molecule in a sample to obtain barcode-tagged molecules: amplifying the barcode-tagged molecules; fragmenting the amplified barcode-tagged molecules to obtain barcode-containing fragments; juxtaposing the barcode-containing fragments to random short segments of the original DNA template molecule during the process of generating a sequencing library to obtain demultiplexed reads; and assembling the demultiplexed reads to obtain extended sequence reads for each DNA template molecule, is disclosed.
1 . A method comprising:
(i) assigning a specific barcode sequence to template DNA molecules in a sample, wherein the assigning comprises a polymerase chain reaction (PCR) amplification reaction using primers comprising universal sequences at the 5′ ends of the primers, and wherein individual primers additionally comprise barcodes, thereby generating barcode-tagged molecules;
(ii) clonally amplifying the barcode-tagged molecules, thereby generating amplified barcode-tagged molecules;
(iii) randomly fragmenting the amplified barcode-tagged molecules using a mechanical method or an enzymatic method, thereby obtaining barcode-containing fragments with barcode-tagged ends and barcode-distal ends;
(iv) circularizing the barcode-containing fragments by intramolecular ligation, thereby juxtaposing the barcode-tagged ends of the barcode-containing fragments with the barcode-distal ends of the barcode-containing fragments and generating a sequencing library of overlapping fragments, wherein the sequencing library is compatible for sequencing on a massively parallel sequencing platform with a maximum read length of around 250 basepairs;
(v) obtaining demultiplexed reads from the sequencing library, wherein demultiplexed reads from the sequencing library comprise sequences of the barcode and the barcode-distal ends of the barcode-containing fragments; and
(vi) assembling the demultiplexed reads to obtain extended sequence reads for the template DNA molecules.
2 . The method of claim 1 , comprising protecting barcode-proximal ends of the amplified barcode-tagged molecules, thereby generating protected barcode-tagged molecules after step (ii).
3 . The method of claim 1 , further comprising labelling the amplified barcode-tagged molecules with biotin.
4 . The method of claim 3 , further comprising purifying biotin-labelled barcode-containing fragments using streptavidin-coated paramagnetic beads.
5 . The method of claim 1 , wherein the barcode-containing fragments comprise between about 300 base pairs to N base pairs, wherein N equals a length of one of the template DNA molecules.
6 . The method of claim 1 , wherein clonally amplifying the barcode-tagged molecules comprises circularizing the barcode-tagged molecules and performing rolling circle amplification.
7 . The method of claim 6 , wherein the rolling circle amplification is performed using a phi29 polymerase.
8 . The method of claim 1 , further comprising ligating sequencing adaptors to the barcode-containing fragments prior to the circularizing.
9 . The method of claim 1 , wherein the template DNA molecules comprise a population of mixed template DNA molecules.
10 . The method of claim 1 , wherein the sequencing library comprises double-stranded DNA circularized molecules.
11 . The method of claim 1 , wherein the template DNA molecules have a length of at least 3 kilobases (kb).
12 . The method of claim 1 , wherein the circularizing is performed by intramolecular ligation using a DNA ligase.
13 . The method of claim 1 , wherein:
(a) the mechanical method comprises focused ultrasound; or
(b) the enzymatic method comprises a dsDNA Fragmentase.
14 . A method comprising:
(i) assigning a specific barcode sequence to template DNA molecules in a sample, wherein the assigning comprises a polymerase chain reaction (PCR) amplification reaction using primers comprising universal sequences at the 5′ ends of the primers, and wherein individual primers additionally comprise barcodes, thereby generating barcode-tagged molecules;
(ii) clonally amplifying the barcode-tagged molecules, thereby generating amplified barcode-tagged molecules;
(iii) fragmenting the amplified barcode-tagged molecules comprising:
(a) creating nicks at the barcode-distal ends of the amplified barcode-tagged molecules,
(b) performing a nick translation towards the barcode-proximal ends of the amplified barcode-tagged molecules, and
(c) treating the resulting molecules with an endonuclease that generates blunt ends, thereby obtaining barcode-containing fragments;
(iv) circularizing the barcode-containing fragments by intramolecular ligation, thereby juxtaposing a barcode-tagged end of the barcode-containing fragments with a barcode-distal end of the barcode-containing fragments and generating a sequencing library of overlapping fragments, wherein the sequencing library is compatible for sequencing on a massively parallel sequencing platform with a maximum read length of around 250 basepairs;
(v) obtaining demultiplexed reads from the sequencing library, wherein demultiplexed reads from the sequencing library comprise sequences of the barcode and the barcode-distal ends of the barcode-containing fragments; and
(vi) assembling the demultiplexed reads to obtain extended sequence reads for the template DNA molecules.
15 . The method of claim 14 , comprising protecting barcode-proximal ends of the amplified barcode-tagged molecules, thereby generating protected barcode-tagged molecules after step (ii).
16 . The method of claim 14 , further comprising labelling the amplified barcode-tagged molecules with biotin.
17 . The method of claim 16 , further comprising purifying biotin-labelled barcode-containing fragments using streptavidin-coated paramagnetic beads.
18 . The method of claim 14 , wherein the barcode-containing fragments comprise between about 300 base pairs to N base pairs, wherein N equals a length of one of the template DNA molecules.
19 . The method of claim 14 , wherein clonally amplifying the barcode-tagged molecules comprises circularizing the barcode-tagged molecules and performing rolling circle amplification.
20 . The method of claim 19 , wherein the rolling circle amplification is performed using a phi29 polymerase.
21 . The method of claim 14 , further comprising ligating sequencing adaptors to the barcode-containing fragments prior to the circularizing.
22 . The method of claim 14 , wherein the template DNA molecules comprise a population of mixed template DNA molecules.
23 . The method of claim 14 , wherein the sequencing library comprises double-stranded DNA circularized molecules.
24 . The method of claim 14 , wherein the template DNA molecules have a length of at least 3 kilobases (kb).
25 . The method of claim 14 , wherein the circularizing is performed by intramolecular ligation using a DNA ligase.