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Botond Sipos

Publications and source records attributed to Botond Sipos.

2 recordsLinked to original sources

Highly parallel direct RNA sequencing on an array of nanopores

Ribonucleic acid sequencing can allow us to monitor the RNAs present in a sample. This enables us to detect the presence and nucleotide sequence of viruses, or to build a picture of how active transcriptional processes are changing - information that is useful for understanding the status and function of a sample. Oxford Nanopore Technologies sequencing technology is capable of electronically analysing a samples DNA directly, and in real-time. In this manuscript we demonstrate the ability of an array of nanopores to sequence RNA directly, and we apply it to a range of biological situations. Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. There are several potential advantages of this approach over other RNA-seq strategies, including the absence of amplification and reverse transcription biases, the ability to detect nucleotide analogues and the ability to generate full-length, strand-specific RNA sequences. Direct RNA sequencing is a completely new way of analysing the sequence of RNA samples and it will improve the ease and speed of RNA analysis, while yielding richer biological information.

Genomics

PASP - a whole-transcriptome poly(A) tail length determination assay for the Illumina platform

The poly(A) tail, co-transcriptionally added to most eukaryotic RNAs, plays an important role in post-transcriptional regulation through modulating mRNA stability and translational efficiency. The length of the poly(A) tail is dynamic, decreasing or increasing in response to various stimuli through the action of enzymatic complexes, and changes in tail length are exploited in regulatory pathways implicated in various biological processes.\n\nTo date, assessment of poly(A) tail length has mostly relied on protocols targeting only a few transcripts. We present PASP ( poly(A) tail sequencing protocol), a whole-transcriptome approach to measure tail lengths -- including a computational pipeline implementing all necessary analyses. PASP uses direct Illumina sequencing of cDNA fragments obtained through G-tailing of poly(A)-selected mRNA followed by fragmentation and reverse transcription.\n\nAnalysis of reads corresponding to spike-in poly(A) tracts of known length indicated that mean tail lengths can be confidently measured, given sufficient coverage. We further explored the utility of our approach by comparing tail lengths estimated from wild type and {Delta}ccr4-1/pan2 mutant yeasts. The yeast whole-transcriptome tail length distributions showed high consistency between biological replicates, and the expected upward shift in tail lengths in the mutant samples was detected. This suggests that PASP is suitable for the assessment of global polyadenylation status in yeast.\n\nThe correlation of per-transcript mean tail lengths between biological and technical replicates was low (higher between mutant samples). Both, however, reached high values after filtering for transcripts with greater coverage. We also compare our results with those of other methods. We identify a number of improvements that could be used in future PASP experiments and, based on our results, believe that direct sequencing of poly(A) tails can become the method of choice for studying polyadenylation using the Illumina platform

Molecular Biology