# 7.2.RNA Structure ## 1) Probing RNA secondary structure ![](https://4115668567-files.gitbook.io/~/files/v0/b/gitbook-legacy-files/o/assets%2F-LPVsf5VZbQ7h14X29qW%2F-Ll_k_6BUSz-qu8y41Lk%2F-Ll_kaKm0OdYp7Momfuj%2Fshapemap.png?generation=1565075335942888\&alt=media) The probing methods of RNA secondary structure includes icSHAPE, SHAPEmap, DMS, etc. For instance, RNA is treated with a SHAPE reagent that reacts at conformationally dynamic nucleotides. During reverse transcription, polymerase reads through chemical adducts in the RNA and incorporates a nucleotide noncomplementary to the original sequence (red) into the cDNA. The resulting cDNA is sequenced using any massively parallel approach to create a mutational profile. Sequencing reads are aligned to a reference sequence, and nucleotide-resolution mutation rates are calculated, corrected for background and normalized, producing a standard SHAPE reactivity profile. SHAPE reactivities can then be used to model secondary structures, visualize competing and alternative structures, or quantify any process or function that modulates local nucleotide RNA dynamics. ## 2) Running steps (shapemapper) 请首先启动相应 [Docker](https://book.ncrnalab.org/teaching/6.rna-regulation#files)，进入工作目录： ```bash cd /home/test/rna_regulation/structure_seq cd example_data ``` ShapeMapper automates the calculation of RNA structure probing reactivities from mutational profiling (MaP) experiments, in which chemical adducts on RNA are detected as internal mutations in cDNA through reverse transcription and read out by massively parallel sequencing. download scripts and example data from: ``` shapemapper \ --target TPP.fa \ --name "example-results" \ --overwrite \ --min-depth 4000 \ --modified --folder TPPplus \ --untreated --folder TPPminus \ --denatured --folder TPPdenat ``` ### 2a) input 1. TPPplus: modified fasta file 2. TPPminus: untreated fasta file ### 2b) output * example-results\_TPP\_histograms.pdf是数据突变率，read depth的可视化展示。 * example-results\_TPP\_profiles.pdf是数据结果的可视化展示，可以用于质控。 * example-results\_TPP.shape ``` 1 -999 2 -999 3 -999 ``` explanation for each column: ``` column1: nucleotide number column2: reactivity after normalization ``` * example-results\_TPP.map ``` 1 -999 0 G 2 -999 0 G 3 -999 0 C ``` explanation for each column: ``` column1: nucleotide number column2: reactivity after normalization column3: standard error column4: nucleotide sequence ``` * example-results\_TPP\_profile.txt是数据的详细输出 ``` Nucleotide Sequence Modified_mutations Modified_read_depth Modified_effective_depthModified_rate Untreated_mutations Untreated_read_depth Untreated_effective_depth Untreated_rate Denatured_mutations Denatured_read_depth Denatured_effective_depth Denatured_rate Reactivity_profile Std_err HQ_profile HQ_stderr Norm_profile Norm_stderr 1 g 0 4788 4335 0.000000 0 5206 4666 0.000000 0 nan 0.000000 0.000000 nan nan nan nan 2 g 0 4837 3405 0.000000 0 5270 3643 0.000000 0 nan 0.000000 0.000000 nan nan nan nan ``` ### 3) 补充知识 shapemapper2是根据输入的modified和untreated的fasta文件进行计算的。如果fasta文件有多行则在输出时会每行对应一组输出文件。实际使用的计算过程中通常是针对多个转录本的。 ## 4) Homework * 简述structure-seq的原理，查阅文献解释reactivity的含义。