Detection and quantification of sample contamination in immune repertoire analysis
First Claim
1. A method of determining carry over contamination in a sample containing T cells and/or B cells in vitro, comprising:
- determining a clonotype profile for each of a plurality of samples by the following steps;
(i) attaching sequence tags to molecules of recombined nucleic acids of T-cell receptor genes or immunoglobulin genes of the T-cells and/or B-cells to form tag-molecule conjugates, wherein substantially every molecule of the tag-molecule conjugates has a unique sequence tag;
(ii) amplifying the tag-molecule conjugates;
(iii) sequencing the tag-molecule conjugates to produce sequence reads that each comprise a sequence tag portion and a clonotype portion; and
(iv) aligning like sequence tag portions of the sequence reads to determine a clonotype sequence from corresponding clonotype portions of the aligned sequence reads and generating a clonotype profile from the clonotype portions; and
recording nucleotide sequences of the sequence tags of each measurement of a clonotype profile; and
determining carry over contamination in the sample by the presence, absence and/or level of sequence tags from any clonotype profile of said plurality of samples.
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Abstract
The invention is directed to methods for detecting and quantifying nucleic acid contamination in a tissue sample of an individual containing T cells and/or B cells, which is used for generating a sequence-based clonotype profile. In one aspect, the invention is implemented by measuring the presence and/or level of an endogenous or exogenous nucleic acid tag by which nucleic acid from an intended individual can be distinguished from that of unintended individuals. Endogenous tags include genetic identity markers, such as short tandem repeats, rare clonotypes or the like, and exogenous tags include sequence tags employed to determine clonotype sequences from sequence reads.
115 Citations
8 Claims
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1. A method of determining carry over contamination in a sample containing T cells and/or B cells in vitro, comprising:
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determining a clonotype profile for each of a plurality of samples by the following steps; (i) attaching sequence tags to molecules of recombined nucleic acids of T-cell receptor genes or immunoglobulin genes of the T-cells and/or B-cells to form tag-molecule conjugates, wherein substantially every molecule of the tag-molecule conjugates has a unique sequence tag; (ii) amplifying the tag-molecule conjugates; (iii) sequencing the tag-molecule conjugates to produce sequence reads that each comprise a sequence tag portion and a clonotype portion; and (iv) aligning like sequence tag portions of the sequence reads to determine a clonotype sequence from corresponding clonotype portions of the aligned sequence reads and generating a clonotype profile from the clonotype portions; and recording nucleotide sequences of the sequence tags of each measurement of a clonotype profile; and determining carry over contamination in the sample by the presence, absence and/or level of sequence tags from any clonotype profile of said plurality of samples. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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Specification