structure-seq analysis (Shape-map)

background

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.

1.shapemapper

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.

shapemapper \
--target TPP.fa \
--name "example-results" \
--overwrite \
--modified --folder TPPplus \
--untreated --folder TPPminus \
--star-aligner
--verbose
#--denatured --folder TPPdenat

input

  1. TPPplus: modified fasta file
  2. TPPminus: untreated fasta file

    output

  3. example-results_TPP_histograms.pdf
  4. example-results_TPP_profiles.pdf
  5. example-results_TPP.shape 
    1	-999
2	-999
3	-999

    explanation for each column:

    column1: nucleotide number 
column2: reactivity
  6. 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
column3: standard error
column4: nucleotide sequence
  7. example-results_TPP_profile.txt 
    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