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rnaends R package to study exact RNA ends at nucleotide resolution
==================================================================

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The rnaends R package focuses on exact 5'-end and/or 3'-end quantification of RNAs *via* targeted RNA-Seq.

Features
--------

   * `Pre-processing raw reads FASTQ file <vignettes/preprocessing.html>`_
      * Reads parsing and validation
      * Reads feature extraction
      * Demultiplexing into separate fastq files
   * Quantification of 5'-end or 3'-end of mapped reads
      * Unique Molecule Identifiers (UMI) use for PCR duplicates elimination
      * Mapping: `map_fastq <functions/reference_manual.html#map-fastq>`_ wrapper for Bowtie or Subread with appropriate default parameters
      * Quantification: `quantify_5prime <functions/reference_manual.html#quantify-5prime>`_ or `quantify_3prime <functions/reference_manual.html#quantify-3prime>`_ functions
   * Downstream analysis
      * `Transcription start sites (TSS) identification  <vignettes/TSS_identification.html>`_
      * `Signal periodicity analysis  <vignettes/Degradation_profiling.html>`_
      * `Post-transccriptional modifications quantification  <vignettes/Post-transcriptional_modifications.html>`_
      * `Differential proportion statistical analysis  <vignettes/Post-transcriptional_modifications.html>`_


Applications
------------

   * `TSS identification <vignettes/TSS_identification.html>`_
   * `Endoribonucleolytic cleavage sites identification <vignettes/Endoribonucleolytic_sites_prediction.html>`_
   * `Co-translational degradation <vignettes/Degradation_profiling.html>`_
   * `Post-transcriptional modification & differential proportion analysis <vignettes/Post-transcriptional_modifications.html>`_


.. image:: img/rnaends.package.overview.png
   :alt: Overview of the features provided in the rnaends package grouped by phases of an RNA-end sequencing project.

**Figure 1: Overview of the features provided in the rnaends package grouped by
phases of an RNA-end sequencing project.**
**A)** Overview of read structure: 1 random nucleotide, then the 4 nucleotides of
barcode (here either CAAG, GTAT, TACA or TCGG) identifying 4 multiplexed
samples, then the recognition sequence (CGGCACCAACCGAGG), then a UMI (7 random
A, C or G nucleotides), then 3 nucleotides of control sequence (CGC),
and eventually the actual 5' end of the RNA (20 nucleotides) which can be mapped
onto the reference sequence;
**B)** `read_features` table obtained with the functions `init_read_features` and
`add_read_feature` corresponding to the read structure in panel A;
**C)** for FASTQ reads pre-processing and validation based on a read_features table
describing the structure of sequencing reads;
**D)** Distribution of read features validity described in panel A and represented
by an upset plot generated after the parsing, validation and demultiplexing of a
raw read FASTQ file. Each combination of valid features is displayed in the
lower upset plot while the upper bar plot shows the corresponding number of
reads;
**E)** for aligning reads on reference sequences and compute abundances at the
single nucleotide resolution to obtain
**F)** a count table representing the abundances (*count*) of RNA 3' or 5' ends mapped
to a reference sequence (*rname*) at a certain location (*position*) on a strand.
In this example, the reads contain UMI extracted in the pre-processing step that
are used at this step to remove PCR amplification duplicates introduced in the
library preparation;
**G)** `rnaends` provides functions for downstream analysis of the count table for
TSS identification, analysis of RNA-ends species proportions (*e.g.* 5'P/5’PPP
in WT *vs.* mutant strain), periodic signal analysis such as ribosome translation
speed profiling, or the exploration of 3' end modifications;
**H)** rnaends count table can also be analyzed further by other libraries: we
illustrate this by a differential expression analysis to identify
endoribonucleolytic cleavage sites by comparing abundances between WT and
endoRNase deletion mutant strains.


.. toctree::
   :maxdepth: 3
   :caption: Installation

   INSTALL.md
   perf/performances.laptop16GB.md

.. toctree::
   :maxdepth: 3
   :caption: Vignettes

   vignettes/preprocessing.md
   vignettes/TSS_identification.md
   vignettes/Endoribonucleolytic_sites_prediction.md
   vignettes/Degradation_profiling.md
   vignettes/Post-transcriptional_modifications.md


.. toctree::
   :maxdepth: 1
   :caption: Reference manual

   functions/reference_manual.md


.. toctree::
   :maxdepth: 1
   :caption: About

   ABOUT.md