NanoMod

This module allows to predict the loci with RNA modifications starting from data produced by NanoPreprocess.

Workflow

  • index_reference index the reference file for Epinano
  • call_variants uses Samtools for calling the variants for Epinano
  • calc_var_frequencies it uses TSV_to_Variants_Freq.py3 for calculating the frequencies of each variants for Epinano
  • predict_with_EPInano It predicts the modifications with Epinano in parallel splitting the input file in 1 million rows
  • combineEpinanoPred It combine the results from Epinano
  • resquiggling resquiggle fast5 files for Tombo
  • getModifications it estimates the modifications using Tombo comparing WT vs KO

Input Parameters

  1. input_path path to the folders produced by NanoPreprocessing step.
  2. comparison tab separated text file containing the list of comparison. Here an example:
WT1 KO1
WT2 KO2
WT3 KO3
  1. reference reference transcriptome
  2. output folder
  3. coverage read coverage threshold for prediction
  4. tombo_opt options for tombo
  5. epinano_opt options for epinano
  6. email

Results

Three folders are produced by this module:

  1. Epinano, containing the results obtained with this method. You have a single file with putative modifications:
#Kmer,Window,Ref,Coverage,q1,q2,q3,q4,q5,mis1,mis2,mis3,mis4,mis5,ins1,ins2,ins3,ins4,ins5,del1,del2,del3,del4,del5,prediction,dist,ProbM,Pro
bU
AGTGG,394404:394405:394406:394407:394408,chr2,8.0:8.0:7.0:7.0:7.0,21.5,21.25,19.857,23.0,16.285999999999998,0.0,0.0,0.0,0.0,0.0,0.0,0.062,0.0
71,0.0,0.0,0.0,0.0,0.0,0.0,0.0,unm,19.26143361547619,3.00000089999998e-14,0.9999999999999699
TTTTT,12150:12151:12152:12153:12154,chr8,3.0:3.0:3.0:3.0:3.0,0.0,16.5,18.5,16.0,16.0,0.0,0.0,0.0,0.0,0.0,0.0,0.0,0.0,0.0,0.0,1.0,0.3329999999
9999996,0.33299999999999996,0.0,0.0,unm,2.5976484688977424,0.06071658133381308,0.9392834186661868
ACATT,438165:438166:438167:438168:438169,chr13,67.0:67.0:67.0:68.0:68.0,13.635,13.446,9.323,9.6,12.127,0.03,0.045,0.015,0.147,0.0740000000000
0001,0.0,0.0,0.0,0.0,0.0,0.06,0.03,0.075,0.11800000000000001,0.07400000000000001,unm,0.08435556637195174,0.519879422458087,0.4801205775419129
5...

and three plots in pdf indicating possible events related to insertion, deletion and mismatches, see the example below.

  1. Tombo, containing the results obtained with this method in fasta format. You have one file for each comparison WT vs KO
>chr11:455562:- Est. Frac. Alternate: 0.98
TGACA
>chr12:1008723:- Est. Frac. Alternate: 0.98
TATCT
>chr15:491587:+ Est. Frac. Alternate: 0.96
TATAT
>chr10:425794:- Est. Frac. Alternate: 0.95
ATGTT
>chr13:510759:+ Est. Frac. Alternate: 0.95
...

And for convenience a 6 bed files with the coordinates of the event.