Understanding the Challenge in RNA Sequencing
The eukaryotic transcriptome is incredibly complex, with many types of RNA molecules—including messenger RNAs (mRNAs), non-coding RNAs, and circular RNAs—interacting within cells. Researchers often need to identify specific transcripts for diseases or mutations, but traditional RNA sequencing methods have their challenges. Most methods rely on complicated steps to enrich target RNAs, which can disrupt the analysis of the entire transcriptome. This makes it difficult to capture a complete picture of what is happening at the molecular level.
What Is PROFIT-seq?
Researchers at the Chinese Academy of Sciences have developed a new method called PROFIT-seq (Programmable Full-Length Isoform Transcriptome Sequencing) that offers a solution. It enriches specific transcripts while preserving the ability to measure the whole transcriptome. Unlike conventional techniques that risk losing important information, PROFIT-seq strikes a balance—it focuses on the RNAs researchers care about most while still giving insight into the broader RNA landscape.
How Does PROFIT-seq Work?
PROFIT-seq works by:
Combinatorial Reverse Transcription: This process captures multiple RNA types, including polyadenylated, non-polyadenylated, and circular RNAs, ensuring comprehensive RNA analysis.
Programmable Control System: During sequencing, the system selectively enriches the most relevant target RNAs—such as transcripts related to specific pathogens or genetic mutations.
Real-time and programmable transcriptome sequencing with PROFIT-seq
a, A schematic overview of the PROFIT-seq method. First, the ribosomal RNA-depleted total RNA was reverse transcribed using a combinatorial RT strategy. Double-stranded oligo(dT) (dsdT), dsN and ssN were successively added to capture polyadenylated, non-polyadenylated and circular RNAs. The reverse-transcribed cDNA was then circularized and amplified using the RCA assay. The nanopore sequencing library was constructed, and PROFIT-seq was used for real-time control of the sequencing process. The acquired chunk data were basecalled and demultiplexed according to the sequencing time, channel number and detected barcodes. The basecalled sequences were subsequently aligned to the reference genome. Finally, PROFIT-seq determined whether the sequencing process should be continued or rejected according to the user-provided sequencing configuration. b, The length of rejected (purple) and finished (green) reads for canonical DNA, cDNA and PROFIT-seq runs. All bulk fast5 runs were simulated for sequencing all reads or rejecting all reads for 1 h. c, The elapsed time for raw signal basecalling, sequence alignment and pore manipulation for each acquired chunk of data. d, PROFIT-seq provides three manipulation modes, including enrichment or depletion of reads aligned to target regions and the balancing mode for dynamic determination of enriching targets based on the accomplished coverage. e, The performance of enriching chromosomes 1, 2, 5, 11 and 12 and depleting other chromosomes (middle) or balancing coverage of all chromosomes (right).
This innovative approach increases data efficiency by over three times and cuts the time needed to detect critical mutations or pathogens by 75%.
A Real-World Application: Colorectal Polyp Development
The researchers demonstrated the power of PROFIT-seq by studying the development of colorectal polyps. They uncovered complex interactions between the host’s immune response and bacterial infections, highlighting the potential role of microbes in driving polyp formation. This detailed insight was only possible because PROFIT-seq allowed them to analyze both targeted transcripts and the full transcriptome at the same time.
Why PROFIT-seq is Important
PROFIT-seq is a breakthrough tool for clinical research and diagnostics. It offers:
Efficient Target Enrichment: Faster identification of pathogens and key mutations.
Comprehensive Transcriptome Coverage: Avoids the trade-off between enrichment and unbiased quantification.
Broad Applications: Useful in clinical diagnostics, targeted research, and understanding disease mechanisms.
By improving both speed and accuracy, PROFIT-seq opens up new possibilities for disease research and personalized medicine. This technology could transform how we diagnose infections, study genetic diseases, and develop new treatments by giving researchers the best of both worlds: focused insights without sacrificing a complete molecular view.