Amplicon-based sequencing using dna spike-ins
AssigneesThe Broad Institute, Inc., President and Fellows of Harvard College
InventorsPardis Sabeti, Bronwyn Macinnis, Erica Normandin, Katie Siddle, Steven Reilly, Matthew Bauer, Kim Lagerborg
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Abstract
Embodiments disclosed herein provide methods of using synthetic DNA spike-ins (SDSIs) to detect, prevent, and quantify contamination in amplicon sequencing. These embodiments may, but are not limited to, reveal sample swaps, intra-batch contamination, and, on a larger scale, intra-laboratory contamination. Embodiments disclosed herein also provide synthetic DNA spike-ins for use in amplicon-based sequencing methods.
Claims14
A method for detecting contamination in amplicon sequencing, said method consisting of:
- (a) providing one or more cDNA samples, each of which contains a target sequence;
- (b) adding a synthetic DNA spike-in (SDSI) to each cDNA sample of the one or more cDNA samples, wherein each SDSI is capable of being amplified simultaneously with the target sequence present in the cDNA sample to which the SDSI is added,
- wherein each SDSI comprises
- a unique core nucleotide sequence capable of differentiating each SDSI, covalently linked to an upstream 5′ primer binding region, and a downstream 3′ primer binding region,
- wherein the nucleotide sequence of the 5′ and 3′ primer binding regions is the same in each SDSI;
- (c) amplifying each target sequence present in the one or more cDNA samples together with the unique core sequence present in each added SDSI;
- (d) sequencing the amplified target and SDSI sequences, and
- (e) determining the presence of the amplified SDSI sequence(s) in the one or more cDNA samples,
- wherein detection of a single SDSI unique core sequence in each cDNA sample indicates contamination-free amplification of each cDNA sample, and
- wherein detection of more than one SDSI unique core sequence in a cDNA sample indicates possible contamination of that cDNA sample with cDNA(s) from a different cDNA sample.
A method for detecting contamination in amplicon sequencing comprising:
- (a) providing one or more cDNA samples, each of which contains a target sequence;
- (b) adding a synthetic DNA spike-in (SDSI) to each cDNA sample of the one or more cDNA samples, wherein each SDSI is capable of being amplified simultaneously with the target sequence present in the cDNA sample to which the SDSI is added,
- wherein each SDSI comprises a unique core nucleotide sequence capable of differentiating each SDSI, covalently linked to an upstream 5′ primer binding region and a downstream 3′ primer binding region,
- wherein the nucleotide sequence of the 5′ and 3′ primer binding regions is the same in each SDSI, and
- wherein each SDSI's unique core sequence is chosen from the nucleotide sequences of SEQ ID NOs:
- 1-96 and 193-291,
- (c) amplifying each target sequence present in the one or more cDNA samples together with the unique core sequence present in each added SDSI;
- (d) sequencing the amplified target and SDSI sequences, and
- (e) determining the presence of the amplified SDSI sequence(s) in the one or more cDNA samples,
- wherein detection of a single SDSI unique core sequence in each cDNA sample indicates contamination-free amplification of each cDNA sample, and
- wherein detection of more than one SDSI unique core sequence in a cDNA sample indicates possible contamination of that cDNA sample with cDNA(s) from a different cDNA sample.
Dependencies are inferred from the claim text.