Internal Standard Nucleic Acid for Assembly Quality and GC Bias
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Solution Overview
Problem
Existing genomic and metagenomic analysis methods lack accurate means for evaluating assembly quality and quantifying microbial communities, particularly due to technical biases such as GC content variation and the inability to assess assemblies without single-copy marker genes.
Innovation Solution
Development of artificial nucleic acid sequences with controlled GC content and recognizable markers, allowing evaluation of assembly quality and quantification through tools like CheckM, and enabling rigorous assessment of GC bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If exogenous nucleic acid with sequence not present in sample is used as internal standard, then absolute quantification capability is improved, but assembly quality assessment capability deteriorates
Solution Approach 1:
The internal standard nucleic acid employs local quality by incorporating specific single-copy marker genes (ribosomal proteins, transcription/translation factors) into its structure. These marker genes are strategically placed within the artificial sequence to enable assembly quality assessment, while the overall non-natural sequence maintains its utility for absolute quantification without interfering with native genome analysis.
Solution Approach 2:
The internal standard represents a composite material combining natural biological elements (single-copy marker genes with known sequences) and artificial components (non-natural sequence regions). This composite structure allows the molecule to simultaneously provide assembly quality assessment through recognizable marker genes and absolute quantification through its unique, distinguishable sequence that does not match any native genome.
2Quantity of substance
If standard nucleic acid without single-copy marker genes is used, then quantification capability is improved, but assembly quality evaluation capability deteriorates
Solution Approach 1:
The internal standard nucleic acid is segmented into functional regions: quantification regions with non-natural sequences for abundance measurement, and assessment regions containing single-copy marker genes for quality evaluation. This segmentation allows each function to operate independently and optimally without mutual interference.
Solution Approach 2:
The internal standard achieves universality by being multi-functional: it serves both as a quantification reference (through its known concentration and unique sequence) and as an assembly quality control (through incorporated marker genes). This eliminates the need for separate control mechanisms.
3Reliability
If artificial nucleic acid sequence is used as internal standard, then non-natural sequence distinguishability is improved, but GC bias assessment capability deteriorates
Solution Approach 1:
The internal standard employs parameter changes by incorporating regions with deliberately varied GC content (including extreme values like very high or very low GC regions) into its structure. This allows the same molecule to maintain non-natural sequence distinguishability while simultaneously serving as a probe for detecting GC bias in sequencing and assembly processes.
Data Source
Figure 1
Figure 2A~2D
Figure 3A~3B
AI summary
Provided is a nucleic acid molecule comprising an artificial nucleic acid sequence and/or a complementary sequence thereof, or a partial fragment sequence thereof, the artificial nucleic acid sequence consisting of: (1) one copy each of artificial genes encoding the following non-naturally occurring sequences (a) to (p); (2) artificial intergenic sequences for linking the artificial genes, each independently consisting of a non-naturally occurring random sequence of 10 to 60 nucleotides in length; and (3) a front spacer sequence and an end spacer sequence, each independently consisting of a non-naturally occurring random sequence of 200 to 400 nucleotides in length.