Limits...
RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids.

Sample PJ, Gaston KW, Alfonzo JD, Limbach PA - Nucleic Acids Res. (2015)

Bottom Line: Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers.Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers.There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined 'variable sequencing', which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA Pat.Limbach@uc.edu.

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Sequencing accuracy in relation to oligomer length. (A) Accuracy as a function of RNA oligomer length. (B) Relative rank of all tested oligomers. First 95% (73/77) second 3% (2/77), fifth 1% (1/77) and uninterpreted 1% (1/77).
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Figure 4: Sequencing accuracy in relation to oligomer length. (A) Accuracy as a function of RNA oligomer length. (B) Relative rank of all tested oligomers. First 95% (73/77) second 3% (2/77), fifth 1% (1/77) and uninterpreted 1% (1/77).

Mentions: To examine the limits of oligomers that could be sequenced by RoboOligo, select MS/MS spectra from the RNase T1 digest of L. lactis total tRNA were chosen. Within the LC-MS/MS data, two 14-mers were identified manually, ACUCUU[t6A]AUCUAUGp and ACU[cmnm5s2U]UU[t6A]AUCAAAGp (35). Automated sequencing of the spectra associated with these 14-mers found that the former sequence was correctly identified as the top scorer while the latter sequence scored as the second highest and had a cumulative abundance that differed from the top result by 1%. Another 48 MS/MS spectra from various RNA samples (Supplementary Table S1) were analyzed by RoboOligo in the same manner as the two tRNA isoacceptors described above. Regarded as a whole, the RoboOligo automated de novo sequencing algorithm correctly annotated the sequences of 73 out of 77 (94%) independently verified oligomers. Of the four incorrect sequence annotations, two of these sequences were the second highest scoring choices, one sequence was the fifth highest scoring result and one sequence could not be determined (Figure 4).


RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids.

Sample PJ, Gaston KW, Alfonzo JD, Limbach PA - Nucleic Acids Res. (2015)

Sequencing accuracy in relation to oligomer length. (A) Accuracy as a function of RNA oligomer length. (B) Relative rank of all tested oligomers. First 95% (73/77) second 3% (2/77), fifth 1% (1/77) and uninterpreted 1% (1/77).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446411&req=5

Figure 4: Sequencing accuracy in relation to oligomer length. (A) Accuracy as a function of RNA oligomer length. (B) Relative rank of all tested oligomers. First 95% (73/77) second 3% (2/77), fifth 1% (1/77) and uninterpreted 1% (1/77).
Mentions: To examine the limits of oligomers that could be sequenced by RoboOligo, select MS/MS spectra from the RNase T1 digest of L. lactis total tRNA were chosen. Within the LC-MS/MS data, two 14-mers were identified manually, ACUCUU[t6A]AUCUAUGp and ACU[cmnm5s2U]UU[t6A]AUCAAAGp (35). Automated sequencing of the spectra associated with these 14-mers found that the former sequence was correctly identified as the top scorer while the latter sequence scored as the second highest and had a cumulative abundance that differed from the top result by 1%. Another 48 MS/MS spectra from various RNA samples (Supplementary Table S1) were analyzed by RoboOligo in the same manner as the two tRNA isoacceptors described above. Regarded as a whole, the RoboOligo automated de novo sequencing algorithm correctly annotated the sequences of 73 out of 77 (94%) independently verified oligomers. Of the four incorrect sequence annotations, two of these sequences were the second highest scoring choices, one sequence was the fifth highest scoring result and one sequence could not be determined (Figure 4).

Bottom Line: Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers.Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers.There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined 'variable sequencing', which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA Pat.Limbach@uc.edu.

Show MeSH
Related in: MedlinePlus