Sequence Tag Multiplex Amplification for MRD Monitoring
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Solution Overview
Problem
Current methods for assessing minimal residual disease in cancer patients face challenges such as increased spurious amplifications, biased sequence representation, and contamination issues during large-scale multiplex amplification of immune receptor chains, which hinder the development of efficient single-reaction amplification techniques for monitoring cancer-related nucleic acids.
Innovation Solution
A method involving the attachment of sequence tags to recombined nucleic acid molecules, followed by amplification and sequencing, allows for the alignment and coalescing of sequence reads to determine clonotypes, thereby monitoring minimal residual disease by detecting the presence, absence, and levels of cancer-correlated clonotypes in a patient.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If large-scale multiplex amplification of immune receptor chains is performed, then the ability to assess minimal residual disease is improved, but spurious amplifications and biased sequence representation increase
Solution Approach 1:
The patent divides the amplification process into multiple sequential steps with distinct primer sets. First primers attach sequence tags to target nucleic acids, then second primers amplify the tagged products. This segmentation allows each step to be optimized independently, reducing spurious amplifications while maintaining measurement precision for minimal residual disease assessment.
Solution Approach 2:
Sequence tags serve as intermediary elements between the target immune receptor chains and the amplification process. These tags are attached to targets in a first amplification step, then serve as binding sites for second primers in a subsequent amplification step. This intermediary mechanism enables highly multiplexed amplification while reducing biased sequence representation through controlled primer binding.
2Productivity
If the scale of multiplexing is increased, then more immune receptor chains can be assessed simultaneously, but primer-dimer formation and spurious amplifications increase
Solution Approach 1:
The patent segments the amplification into two distinct phases: a first amplification that attaches sequence tags with minimal priming, and a second amplification that uses the attached tags as templates. This segmentation reduces primer-dimer formation by separating the functions of target attachment and product amplification, enabling higher multiplexing scales with reduced spurious amplifications.
Solution Approach 2:
The first amplification step performs preliminary action by attaching sequence tags to all target nucleic acids before the main amplification occurs. This preliminary tagging ensures that subsequent amplification primers bind only to intended targets rather than forming dimers, allowing increased multiplexing capacity without proportional increases in primer-dimer formation.
3Loss of information
If sequence tags are incorporated into amplified sequences, then sample tracking and contamination detection are improved, but amplification difficulties are exacerbated
Solution Approach 1:
The patent merges the function of amplification with the function of sequence tag attachment by incorporating tags directly into the amplified products during the first amplification step. This combining eliminates separate tagging steps, reducing overall process complexity while maintaining sample tracking capability through the incorporated sequence tags.
Solution Approach 2:
The sequence tags attached during the first amplification step serve dual purposes: they mark samples for tracking and contamination detection, and they simultaneously serve as binding sites for second primers in the subsequent amplification step. This self-service mechanism reduces process complexity by making the tags functional components of the amplification system rather than separate additives.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables efficient large-scale amplification and sequencing of immune receptor chains in a single reaction, reducing contamination and bias, and provides a sensitive method for monitoring minimal residual disease by generating accurate clonotype profiles.
Implementation Method 1
each primer of the first set comprises a receptor-specific portion, a 5'-non-complementary end containing a first primer binding site and a sequence tag disposed between the receptor-specific portion and the first primer binding site, wherein the receptor-specific portion anneals to a different recombined nucleic acid at a predetermined location
Implementation Method 2
combining in a reaction mixture under primer extension conditions a first set of primers with the sample, wherein each primer of the first set comprises a receptor-specific portion... and is extended to form a first extension product
Data Source
Figure 1A
Figure 1B
Figure 1C
AI summary
The invention is directed to sequence-based profiling of populations of nucleic acids by multiplex amplification and attachment of one or more sequence tags to target nucleic acids and/or copies thereof followed by high-throughput sequencing of the amplification product. In some embodiments, the invention includes successive steps of primer extension, removal of unextended primers and addition of new primers either for amplification (for example by PCR) or for additional primer extensions. Some embodiments of the invention are directed to minimal residual disease (MRD) analysis of patients being treated for cancer. Sequence tags incorporated into sequence reads provide an efficient means for determining clonotypes and at the same time provide a convenient means for detecting carry-over contamination from other samples of the same patient or from samples of a different patient which were tested in the same laboratory.