Full-length PacBio Amplicon Sequencing to Unveil RNA Editing Sites


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Abstract

Background:RNA editing enriches post-transcriptional sequence changes. Currently detecting RNA editing sites is mostly based on the Sanger sequencing platform and second-generation sequencing. However, detection with Sanger sequencing is limited by the disturbing background peaks using the direct sequencing method and the clone number using the clone sequencing method, while second-generation sequencing detection is constrained by its short read.

Objective:We aimed to design a pipeline that can accurately detect RNA editing sites for full-length long-read amplicons to meet the requirement when focusing on a few specific genes of interest.

Method:We developed a novel high-throughput RNA editing sites detection pipeline based on the PacBio circular consensus sequences sequencing which is accurate with high-throughput and long-read coverage. We tested the pipeline on cytosolic malate dehydrogenase in the hard-shelled mussel Mytilus coruscus and further validated it using direct Sanger sequencing.

Results:Data generated from the PacBio circular consensus sequences (CCS) amplicons in three mussels were first filtered by quality and then selected by open reading frame. After filtering, 225-2047 sequences of the three mussels, respectively, were used to identify RNA editing sites. With corresponding genomic DNA sequences, we extracted 227-799 candidate RNA editing sites excluding heterozygous sites. We further figured out 7-11 final RESs using a new error model specially designed for RNA editing site detection. The resulting RNA editing sites all agree with the validation using the Sanger sequencing.

Conclusion:We report a near-zero error rate method in identifying RNA editing sites of long-read amplicons with the use of PacBio CCS sequencing.

About the authors

Xiao-Lu Zhu

Key Laboratory of Mariculture of Ministry of Education, College of Fisheries, Ocean University of China

Email: info@benthamscience.net

Ming-Ling Liao

Key Laboratory of Mariculture of Ministry of Education, College of Fisheries, Ocean University of China

Author for correspondence.
Email: info@benthamscience.net

Ya-Jie Zhu

Key Laboratory of Mariculture of Ministry of EducationCollege of Fisheries, College of Fisheries, Ocean University of China

Email: info@benthamscience.net

Yun-Wei Dong

Key Laboratory of Mariculture of Ministry of Educationtion, College of Fisheries, College of Fisheries, Ocean University of China

Email: info@benthamscience.net

References

  1. Picardi E, Manzari C, Mastropasqua F, Aiello I, D’Erchia AM, Pesole G. Profiling RNA editing in human tissues: Towards the inosinome Atlas. Sci Rep 2015; 5(1): 14941. doi: 10.1038/srep14941 PMID: 26449202
  2. Farajollahi S, Maas S. Molecular diversity through RNA editing: A balancing act. Trends Genet 2010; 26(5): 221-30. doi: 10.1016/j.tig.2010.02.001 PMID: 20395010
  3. Gott JM, Emeson RB. Functions and mechanisms of RNA editing. Annu Rev Genet 2000; 34(1): 499-531. doi: 10.1146/annurev.genet.34.1.499 PMID: 11092837
  4. Small ID, Schallenberg-Rüdinger M, Takenaka M, Mireau H, Ostersetzer-Biran O. Plant organellar RNA editing: What 30 years of research has revealed. Plant J 2020; 101(5): 1040-56. doi: 10.1111/tpj.14578 PMID: 31630458
  5. Bar-Yaacov D, Mordret E, Towers R, et al. RNA editing in bacteria recodes multiple proteins and regulates an evolutionarily conserved toxin-antitoxin system. Genome Res 2017; 27(10): 1696-703. doi: 10.1101/gr.222760.117 PMID: 28864459
  6. Chateigner-Boutin AL, Small I. Plant RNA editing. RNA Biol 2010; 7(2): 213-9. doi: 10.4161/rna.7.2.11343 PMID: 20473038
  7. Garrett S, Rosenthal JJC. RNA editing underlies temperature adaptation in K+ channels from polar octopuses. Science 2012; 335(6070): 848-51. doi: 10.1126/science.1212795 PMID: 22223739
  8. Hwang T, Park CK, Leung AKL, et al. Dynamic regulation of RNA editing in human brain development and disease. Nat Neurosci 2016; 19(8): 1093-9. doi: 10.1038/nn.4337 PMID: 27348216
  9. Niavarani A, Currie E, Reyal Y, et al. APOBEC3A is implicated in a novel class of G-to-A mRNA editing in WT1 transcripts. PLoS One 2015; 10(3): e0120089. doi: 10.1371/journal.pone.0120089 PMID: 25807502
  10. Nishikura K. Functions and regulation of RNA editing by ADAR deaminases. Annu Rev Biochem 2010; 79(1): 321-49. doi: 10.1146/annurev-biochem-060208-105251 PMID: 20192758
  11. Hsiao YHE, Bahn JH, Yang Y, et al. RNA editing in nascent RNA affects pre-mRNA splicing. Genome Res 2018; 28(6): 812-23. doi: 10.1101/gr.231209.117 PMID: 29724793
  12. Solomon O, Di Segni A, Cesarkas K, et al. RNA editing by ADAR1 leads to context-dependent transcriptome-wide changes in RNA secondary structure. Nat Commun 2017; 8(1): 1440. doi: 10.1038/s41467-017-01458-8 PMID: 29129909
  13. Song B, Shiromoto Y, Minakuchi M, Nishikura K. The role of RNA editing enzyme ADAR1 in human disease. Wiley Interdiscip Rev RNA 2022; 13(1): e1665. doi: 10.1002/wrna.1665 PMID: 34105255
  14. Zhang F, Saha S, Shabalina SA, Kashina A. Differential arginylation of actin isoforms is regulated by coding sequence-dependent degradation. Science 2010; 329(5998): 1534-7. doi: 10.1126/science.1191701 PMID: 20847274
  15. Eisenberg E, Levanon EY. A-to-I RNA editing — immune protector and transcriptome diversifier. Nat Rev Genet 2018; 19(8): 473-90. doi: 10.1038/s41576-018-0006-1 PMID: 29692414
  16. Gallo A, Vukic D, Michalík D, O’Connell MA, Keegan LP. ADAR RNA editing in human disease; more to it than meets the I. Hum Genet 2017; 136(9): 1265-78. doi: 10.1007/s00439-017-1837-0 PMID: 28913566
  17. Garrett SC, Rosenthal JJC. A role for A-to-I RNA editing in temperature adaptation. Physiology 2012; 27(6): 362-9. doi: 10.1152/physiol.00029.2012 PMID: 23223630
  18. Blazej RG, Kumaresan P, Mathies RA. Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. Proc Natl Acad Sci 2006; 103(19): 7240-5. doi: 10.1073/pnas.0602476103 PMID: 16648246
  19. Ishige T, Itoga S, Matsushita K, Nomura F. Locked nucleic acid probe enhances Sanger sequencing sensitivity and improves diagnostic accuracy of high-resolution melting-based KRAS mutational analysis. Clin Chim Acta 2016; 457: 75-80. doi: 10.1016/j.cca.2016.04.005 PMID: 27071699
  20. Sharma S, Patnaik SK, Kemer Z, Baysal BE. Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes. RNA Biol 2017; 14(5): 603-10. doi: 10.1080/15476286.2016.1184387 PMID: 27149507
  21. Callahan BJ, Wong J, Heiner C, et al. High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution. Nucleic Acids Res 2019; 47(18): e103. doi: 10.1093/nar/gkz569 PMID: 31269198
  22. Manyana S, Gounder L, Pillay M, Manasa J, Naidoo K, Chimukangara B. HIV-1 drug resistance genotyping in resource limited settings: Current and future perspectives in sequencing technologies. Viruses 2021; 13(6): 1125. doi: 10.3390/v13061125 PMID: 34208165
  23. Zhang R, Li X, Ramaswami G, et al. Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing. Nat Methods 2014; 11(1): 51-4. doi: 10.1038/nmeth.2736 PMID: 24270603
  24. Sinnamon JR, Kim SY, Fisk JR, et al. In vivo repair of a protein underlying a neurological disorder by programmable RNA editing. Cell Rep 2020; 32(2): 107878. doi: 10.1016/j.celrep.2020.107878 PMID: 32668243
  25. Schadt EE, Turner S, Kasarskis A. A window into third-generation sequencing. Hum Mol Genet 2010; 19(R2): R227-40. doi: 10.1093/hmg/ddq416 PMID: 20858600
  26. Kanwar N, Blanco C, Chen IA, Seelig B. PacBio sequencing output increased through uniform and directional fivefold concatenation. Sci Rep 2021; 11(1): 18065. doi: 10.1038/s41598-021-96829-z PMID: 34508117
  27. Wang Z, Jimenez-Fernandez O, Osenbrück K, et al. Streambed microbial communities in the transition zone between groundwater and a first-order stream as impacted by bidirectional water exchange. Water Res 2022; 217: 118334. doi: 10.1016/j.watres.2022.118334 PMID: 35397370
  28. Liu Z, Quinones-Valdez G, Fu T, Choudhury M, Reese F, Mortazavi A, et al. L-GIREMI uncovers RNA editing sites in long-read RNA-seq. bioRxiv 2022; 2022; 485515. doi: 10.1101/2022.03.23.485515
  29. Wenger AM, Peluso P, Rowell WJ, et al. Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nat Biotechnol 2019; 37(10): 1155-62. doi: 10.1038/s41587-019-0217-9 PMID: 31406327
  30. Wagner J, Coupland P, Browne HP, Lawley TD, Francis SC, Parkhill J. Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification. BMC Microbiol 2016; 16(1): 274. doi: 10.1186/s12866-016-0891-4 PMID: 27842515
  31. Zhang F, Lu Y, Yan S, Xing Q, Tian W. SPRINT: An SNP-free toolkit for identifying RNA editing sites. Bioinformatics 2017; 33(22): 3538-48. doi: 10.1093/bioinformatics/btx473 PMID: 29036410
  32. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 2016; 13(7): 581-3. doi: 10.1038/nmeth.3869 PMID: 27214047
  33. Prodan A, Tremaroli V, Brolin H, Zwinderman AH, Nieuwdorp M, Levin E. Comparing bioinformatic pipelines for microbial 16S rRNA amplicon sequencing. PLoS One 2020; 15(1): e0227434. doi: 10.1371/journal.pone.0227434 PMID: 31945086
  34. Team RCR. A language and environment for statistical computing. R foundation for statistical computing. 2022. Available from: https://www.gbif.org/tool/81287/r-a-language-and-environment-for-statistical-computing
  35. Ding M, Wang Z, Dong Y. Food availability on the shore: Linking epilithic and planktonic microalgae to the food ingested by two intertidal gastropods. Mar Environ Res 2018; 136: 71-7. doi: 10.1016/j.marenvres.2018.02.005 PMID: 29478767
  36. Biosciences P. SMRT®Tools reference guide. 2019. Available from: https://www.pacb.com/wp-content/uploads/SMRT_Tools_Reference_Guide_v11.0.pdf
  37. Haas BJ. 2012. Available from: https://github.com/TransDecoder/TransDecoder
  38. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215(3): 403-10. doi: 10.1016/S0022-2836(05)80360-2 PMID: 2231712
  39. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol 2013; 30(4): 772-80. doi: 10.1093/molbev/mst010 PMID: 23329690
  40. Katoh K, Toh H. Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 2008; 9(4): 286-98. doi: 10.1093/bib/bbn013 PMID: 18372315
  41. Lutzoni F, Wagner P, Reeb V, Zoller S. Integrating ambiguously aligned regions of DNA sequences in phylogenetic analyses without violating positional homology. Syst Biol 2000; 49(4): 628-51. doi: 10.1080/106351500750049743 PMID: 12116431
  42. Zeng Y, Li J, Li G, et al. Correction of the marfan syndrome pathogenic FBN1 mutation by base editing in human cells and heterozygous embryos. Mol Ther 2018; 26(11): 2631-7. doi: 10.1016/j.ymthe.2018.08.007 PMID: 30166242
  43. Yablonovitch AL, Fu J, Li K, et al. Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates. Nat Commun 2017; 8(1): 1570. doi: 10.1038/s41467-017-01658-2 PMID: 29146998
  44. Li Q, Gloudemans MJ, Geisinger JM, et al. RNA editing underlies genetic risk of common inflammatory diseases. Nature 2022; 608(7923): 569-77. doi: 10.1038/s41586-022-05052-x PMID: 35922514
  45. Malik TN, Cartailler J-P, Emeson RB. RNA Editing Methods in Molecular Biology. Berlin: Springer 2021; pp. 97-111.
  46. Nakahama T, Kato Y, Kim JI, et al. ADAR 1‐mediated RNA editing is required for thymic self‐tolerance and inhibition of autoimmunity. EMBO Rep 2018; 19(12): e46303. doi: 10.15252/embr.201846303 PMID: 30361393
  47. Rees HA, Wilson C, Doman JL, Liu DR. Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 2019; 5(5): eaax5717. doi: 10.1126/sciadv.aax5717 PMID: 31086823
  48. Tan MH, Li Q, Shanmugam R, et al. Dynamic landscape and regulation of RNA editing in mammals. Nature 2017; 550(7675): 249-54. doi: 10.1038/nature24041 PMID: 29022589
  49. Diroma MA, Ciaccia L, Pesole G, Picardi E. Elucidating the editome: Bioinformatics approaches for RNA editing detection. Brief Bioinform 2019; 20(2): 436-47. doi: 10.1093/bib/bbx129 PMID: 29040360
  50. Guo Y, Yu H, Samuels DC, Yue W, Ness S, Zhao Y. Single-nucleotide variants in human RNA: RNA editing and beyond. Brief Funct Genomics 2019; 18(1): 30-9. doi: 10.1093/bfgp/ely032 PMID: 30312373
  51. Liu YX, Qin Y, Chen T, et al. A practical guide to amplicon and metagenomic analysis of microbiome data. Protein Cell 2021; 12(5): 315-30. doi: 10.1007/s13238-020-00724-8 PMID: 32394199
  52. Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 2019; 37(8): 907-15. doi: 10.1038/s41587-019-0201-4 PMID: 31375807
  53. Löytynoja A, Goldman N. Phylogeny-aware gap placement prevents errors in sequence alignment and evolutionary analysis. Science 2008; 320(5883): 1632-5. doi: 10.1126/science.1158395 PMID: 18566285
  54. Gerasimov ES, Gasparyan AA, Kaurov I, et al. Trypanosomatid mitochondrial RNA editing: Dramatically complex transcript repertoires revealed with a dedicated mapping tool. Nucleic Acids Res 2018; 46(2): 765-81. doi: 10.1093/nar/gkx1202 PMID: 29220521
  55. Lo Giudice C, Hernández I, Ceci LR, Pesole G, Picardi E. RNA editing in plants: A comprehensive survey of bioinformatics tools and databases. Plant Physiol Biochem 2019; 137: 53-61. doi: 10.1016/j.plaphy.2019.02.001 PMID: 30738217
  56. Deng P, Khan A, Jacobson D, et al. Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila. Nat Commun 2020; 11(1): 1580. doi: 10.1038/s41467-020-15435-1 PMID: 32221286
  57. Zhang Q, Xiao X. Genome sequence–independent identification of RNA editing sites. Nat Methods 2015; 12(4): 347-50. doi: 10.1038/nmeth.3314 PMID: 25730491
  58. da Fonseca RR, Albrechtsen A, Themudo GE, et al. Next-generation biology: Sequencing and data analysis approaches for non-model organisms. Mar Genomics 2016; 30: 3-13. doi: 10.1016/j.margen.2016.04.012 PMID: 27184710

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