Methods for identification of samples
The present invention describes methods, carriers, and vectors relating to nucleic acids for labelling an item, wherein each carrier comprises at least 2 nucleotide barcode nucleic acids, other than sample nucleic acid, for labelling, wherein each nucleotide barcode nucleic acid comprises a different minimal nucleotide barcode sequence with a length of at least 4 nucleotides, where at least two of said different nucleotide barcode nucleic acids have a minimal nucleotide barcode sequence of the same length, wherein the combination of these different nucleotide barcode nucleic acids generates a transferable molecular identification barcode, whereby each such transferable molecular identification barcode is different for each of the carriers in the collection.
1. A method of labelling and identifying a plurality of biological samples comprising one or more target sequences of one or more nucleic acids, the method comprising:
(a) mixing each biological sample from the plurality of biological samples with a unique transferable molecular identification barcode (TMIB) in a carrier from a plurality of carriers at a first time point to form a batch of biological samples, the TMIB comprising a unique combination of at least 2 molecular identification barcodes, wherein said at least 2 molecular identification barcodes do not hybridize to the one or more nucleic acids of the biological samples and are not incorporated in the one or more nucleic acids of the biological sample, wherein each molecular identification barcode comprises:
(i) a unique minimal nucleotide barcode (MNB) with a length of at least 4 nucleotides, and
(ii) a constant sequence flanking the MNB at one or both ends, wherein the constant sequence is not encoded in any naturally occurring genome or cloning vector, and wherein the constant sequence comprises:
an extracting sequence(ES), wherein the ES comprises the same sequence for each TMIB in the batch of biological samples, and wherein the ES is positioned at a defined length from the MNB; and
an identifier sequence (IS), wherein the IS identifies the MNB;
wherein each carrier of the plurality of carriers comprises a substrate or a container, and an exterior of each carrier comprises a macroscopic barcode label (MBL) associated with the corresponding TMIB;
(b) mixing each sample from the batch of biological samples with a unique sample specific pooling barcode (SSPB) in an additional carrier from a plurality of additional carriers at a second time point, wherein the second time point is different from the first time point;
(c) incorporating each SSPB into the TMIB and the target sequences to create a plurality of dual-labelled samples;
(d) pooling the dual-labelled samples;
(e) sequencing the pooled dual-labelled samples by a parallel sequencing method to obtain sequence data comprising:
(i) the one or more target sequences in the biological sample,
(ii) the MNBs, and
(iii) the SSPBs;
(f) grouping the obtained sequence data according to the SSPBs, wherein each group has a different SSPB;
(g) identifying the TMIB for each group of obtained sequence data by identifying a sequence between two extracting sequences at the defined length from the MNB or adjacent to one extracting sequence at the defined length from the MNB;
(h) cross-referencing the identified TMIB to classify each group of obtained sequence data as derived from an accurate sample, a sample switch, and/or a contamination of a sample with and an expected TMIB, wherein the expected TMIB corresponds to the MBL, and wherein:
(i) a sample is accurate and properly processed between the first and second time points when the identified TMIB is identical to the expected TMIB,
(ii) a sample switch has occurred between the first and second time points when the identified TMIB is not identical to the expected TMIB, or
(iii) contamination of a sample has occurred between the first and second time points when more than one TMIB is identified, including the expected TMIB.
2. The method according to claim 1 , wherein incorporating the SSPB into the TMIB and the target sequence comprises ligating adaptors comprising the SSPB to the target sequence, thereby generating ligated products.
3. The method according to claim 2 , further comprising an enrichment step, the enrichment step comprising:
generating and/or isolating the sequences of the ligated products through amplification or capture of the ligated products.
4. The method according to claim 3 , wherein amplification is selected from 1-step PCR, 2-step PCR, primer extension followed by ligation and PCR, or circularisation based amplification.
5. The method according to claim 1 , wherein each TMIB comprises at least two pairs of molecular identification barcodes.
6. The method according to claim 1 , wherein the constant sequence is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:20.
7. The method according to claim 1 , wherein the constant sequence is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:11.
8. The method according to claim 1 , wherein the constant sequence is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:11.
9. The method according to claim 1 , wherein incorporating the SSPB into the TMIB and the target sequence comprises oligonucleotide synthesis using one or more primers comprising the SSPB.
10. The method of claim 1 , wherein the IS comprises the same sequence for each TMIB in the batch of biological samples.