Method for identifying the source of an amplicon
The present invention relates to a method for identifying the source of an amplicon, comprising: providing a plurality of pools of amplicons from different sources, wherein the amplicons from different sources are present in more than one pool, and wherein the amplicons in each pool are tagged with a unique pool-specific identifier; sequencing at least part of the amplicons that comprise the pool-specific identifiers; and assigning one or more of the amplicons to corresponding pools and/or sources using the pool-specific identifiers.
1. A method for identifying the source of an amplicon, comprising:
(a) providing a plurality of pools of amplicons, each pool comprising amplicons from different sources, wherein the amplicons from at least one of the different sources are present in more than one pool, and wherein the amplicons in each pool are tagged with a unique pool-specific identifier;
(b) sequencing at least part of the amplicons that comprise the pool-specific identifiers;
(c) assigning one or more of the amplicons to corresponding pools and/or sources using the pool-specific identifiers.
2. The method according to claim 1 , wherein the sequencing is carried out by high-throughput sequencing.
3. The method according to claim 2 , wherein the high-throughput sequencing is performed on a solid support.
4. The method according to claim 2 , wherein the high-throughput sequencing is based on Sequencing-by-Synthesis.
5. The method according to claim 2 , wherein the high-throughput sequencing comprises the steps of:
annealing the tagged nucleic acid fragments to beads, each bead annealing with a single tagged nucleic acid fragment;
emulsifying the beads in water-in-oil micro reactors, each water-in-oil micro reactor comprising a single bead;
performing emulsion PCR to amplify tagged nucleic acid fragments on the surface of beads,
optionally, selecting and enriching beads comprising amplified tagged nucleic acid fragments;
loading the beads in wells, each well comprising a single bead; and
generating a pyrophosphate signal.
6. The method according to claim 2 , wherein the high-throughput sequencing comprises the steps of:
annealing the tagged nucleic acid fragments to a surface comprising first and second primers or first and second primer binding sequences respectively,
performing bridge amplification to provide clusters of amplified tagged nucleic acid fragments,
determining the nucleotide sequence of the amplified tagged nucleic acid fragments using labelled reversible terminator nucleotides.
7. The method according to claim 1 , wherein the pool-specific identifier is from 4-16 base pairs.
8. The method according to claim 7 , wherein the pool-specific identifier is from 4-10 base pairs.
9. The method according to claim 7 , wherein the pool-specific identifier is from 4-8 base pairs.
10. The method according to claim 7 , wherein the pool-specific identifier is from 4-6 base pairs.
11. The method according to claim 7 , wherein the pool-specific identifier does not comprise two or more identical consecutive bases.
12. The method according to claim 7 , wherein for two or more pools, the corresponding pool-specific identifiers comprise at least two different nucleotides.
13. The method according to claim 1 , wherein the addition of the pool-specific identifier is by ligation of at least one adaptor.
14. The method according to claim 1 , wherein the addition of the pool-specific identifier is by amplification with at least one primer.
15. The method according to claim 1 , wherein step (c) further comprises clustering/aligning the amplicons that comprise identical nucleic acid sequences in part of the amplicons but different pool-specific identifiers.
16. The method according to claim 1 , wherein the amplicons from at least one of the different sources are absent from one or more pools.