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Rare Event Detection Using Error-corrected DNA and RNA Sequencing
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Single molecule counting and assessment of random molecular tagging errors with transposable giga-scale
Billy T Lau1, Hanlee P Ji2,3
1Stanford Genome Technology Center, Stanford University, Palo Alto, CA, 94304, USA.
BMC Genomics
|September 23, 2017
Summary
Transposable error-correcting barcodes accurately identify RNA molecules, overcoming errors common in random-mer barcodes. This improves transcriptome quantification, especially with limited RNA samples.
Area of Science:
- Molecular biology
- Genomics
- Bioinformatics
Background:
- RNA sequencing (RNA-Seq) quantifies gene expression by counting cDNA fragments.
- Molecular barcodes improve RNA-Seq by detecting amplification duplicates but are prone to errors.
- These errors cause inaccurate transcriptome quantification, particularly with low RNA input.
Purpose of the Study:
- To introduce and evaluate transposable error-correcting barcodes for accurate molecular species identification.
- To systematically characterize errors present in random-mer molecular barcodes.
Main Methods:
- Development and application of transposable barcodes for exponential expansion of unique labels.
- Experimental analysis of RNA standards with inline random-mer molecular barcodes.
- Systematic characterization of sequence errors in molecular barcodes.
Main Results:
- Random-mer molecular barcodes exhibit substantial and persistent errors.
- These errors are difficult to resolve and become more dominant at low RNA input amounts.
- Transposable barcodes enable systematic identification of molecular species.
Conclusions:
- This study introduces transposable molecular barcodes for error correction in RNA-Seq.
- Identified extensive errors in random-mer barcodes suggest a need for error-correcting approaches.
- Error-correcting barcodes are recommended for transcriptome analysis, especially with decreasing input amounts.
