Amplicon-based sequencing using dna spike-ins

Patent metadata

Field	Value
Publication number	US-12545952-B2
Application number	US-202217684328-A
Country	US
Kind code	B2
Filing date	Mar 1, 2022
Priority date	Mar 1, 2021
Publication date	Feb 10, 2026
Grant date	Feb 10, 2026

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.

First claim

What is claimed is:

1 . 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.

2 . The method of claim 1 , wherein each SDSI unique core sequence is selected to minimize self-hybridization and cross-hybridization with other nucleic acid molecules present in the one or more cDNA samples.

3 . The method of claim 2 , wherein the sequence homology between each SDSI's unique core sequence and the sequence of the nucleic acid molecules in each cDNA sample is less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 20%, or less than 15%, or less than 5%, or less than 1%, and/or
wherein the length of the sequence homology between each SDSI's unique core sequence and the sequence of the nucleic acid molecules in each cDNA sample is less than 15, or less than 20, or less than 25, or less than 30, or less than 35, or less than 40, or less than 45, or less than 50 contiguous bases, and/or wherein each SDSI is from 50 to 5000 nucleotides in length.

4 . The method of claim 2 , wherein each SDSI's unique core sequence is derived from a rare organism, optionally wherein the rare organism is a thermophilic archaeon.

5 . The method of claim 2 , wherein the unique core sequence of each SDSI comprises a nucleotide sequence chosen from any one of SEQ ID NOs: 1-96 and 193-291, optionally, wherein the unique core sequences comprising the nucleotide sequences of SEQ ID NOs: 16, 57, and 66 are substituted for the nucleotide sequences of SEQ ID NOs: 289, 290, and 291, respectively.

6 . The method of claim 2 , wherein each SDSI comprises the nucleotide sequences chosen from any one of SEQ ID NOs: 97-192 and 292-390, optionally, wherein the unique core sequences comprising the nucleotide sequences of SEQ ID NOs: 112, 153, and 162 are substituted for the nucleotide sequences of SEQ ID NOs: 388, 389, 390, respectively.

7 . The method of claim 2 , wherein the primer binding regions have a Tm between 55-65° C.

8 . The method of claim 2 , wherein the amplification is conducted using SEQ ID NO: 391 as a forward primer and SEQ ID NO: 392 as a reverse primer.

9 . The method of claim 1 , wherein the concentration of each SDSI ranges from 0.1 femtomolar-1.0 femtomolar.

10 . The method of claim 1 , wherein the one or more cDNA samples are for sequencing a pathogen or family of pathogens, optionally, wherein the pathogen is a virus or a bacteria, and the region of the bacterial genome sequenced is associated with antibiotic resistance.

11 . The method of claim 1 , wherein the one or more cDNA samples contain viral nucleic acid sequences.

12 . The method of claim 1 , wherein the one or more cDNA samples are for creating one or more sequencing families/clusters.

13 . The method of claim 1 , wherein the one or more cDNA samples are obtained by reverse transcription of their respective RNA(s).

14 . 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.

Assignees

• Broad Inst Inc
• Harvard College

Inventors

• Sabeti Pardis
• Macinnis Bronwyn
• Normandin Erica
• Siddle Katie
• Reilly Steven
• Bauer Matthew
• Lagerborg Kim

Classifications

• C12Q2527/146

  Concentration of target or template · CPC title

• C12Q2545/101

  with an internal standard/control · CPC title

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q2527/107

  Temperature of melting, i.e. Tm · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title