RiboCode 1.2.4.dev0

Detect translated ORFs using ribosome-profiling data
====================================================

*RiboCode* is a very simple but high-quality computational algorithm to
identify genome-wide translated ORFs using ribosome-profiling data.

Dependencies:
-------------

- pysam

- pyfasta

- h5py

- Biopython

- Numpy

- Scipy

- matplotlib

- setuptools

Installation
------------

*RiboCode* can be installed like any other Python packages. Here are some popular ways:

* Install from PyPI:

.. code-block:: bash

pip install RiboCode

* Install from local:

.. code-block:: bash

pip install RiboCode-*.tar.gz

If you have not administrator permission, you need to install *RiboCode* locally in you own directory by adding the
option ``--user`` to installation commands. Then, you need to add ``~/.local/bin/`` to the ``PATH`` variable,
and ``~/.local/lib/`` to the ``PYTHONPATH`` variable. For example, if you are using the bash shell, you would do
this by adding the following lines to your ``~/.bashrc`` file:

.. code-block:: bash

export PATH=$PATH:$HOME/.local/bin/
export PYTHONPATH=$HOME/.local/lib/python2.7

You then need to source your ``~/.bashrc`` file by this command:

.. code-block:: bash

source ~/.bashrc

Tutorial to analyze ribosome-profiling data and run *RiboCode*
--------------------------------------------------------------

Here, we use the `HEK293 dataset`_ as an example to illustrate the use of *RiboCode*. Please make sure the path of file is correctly.

1. **Required files**

The genome FASTA file, GTF file for annotation can be downloaded from:


http://www.gencodegenes.org

or from:

http://asia.ensembl.org/info/data/ftp/index.html

http://useast.ensembl.org/info/data/ftp/index.html

For example, the required files in this tutorial can be downloaded from following URL:

GTF: ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_19/gencode.v19.annotation.gtf.gz

FASTA: ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_19/GRCh37.p13.genome.fa.gz

The raw Ribo-seq FASTQ file can be download by using fastq-dump tool from `SRA_Toolkit`_:

.. code-block:: bash

fastq-dump -A <SRR1630831>

2. **Trimming adapter sequence for ribo-seq data**

Using cutadapt program https://cutadapt.readthedocs.io/en/stable/installation.html

Example:

.. code-block:: bash

cutadapt -m 20 --match-read-wildcards -a (Adapter sequence) -o <Trimmed fastq file> <Input fastq file>


Here, the adapter sequences for this data had already been trimmed off, so we can skip this step.

3. **Removing ribosomal RNA(rRNA) derived reads**

Align the trimmed reads to rRNA sequences using Bowtie, then select unaligned reads for the next step.

Bowtie program http://bowtie-bio.sourceforge.net/index.shtml

rRNA sequences: We provided a `rRNA.fa`_ file in data folder of this package.

Example:

.. code-block:: bash

bowtie-build <rRNA.fa> rRNA
bowtie -p 8 -norc --un un_aligned.fastq rRNA -q <SRR1630831.fastq> <HEK293_rRNA.align>

4. **Aligning the clean reads to reference genome**

Using STAR program: https://github.com/alexdobin/STAR

Example:

(1). Build index

.. code-block:: bash

STAR --runThreadN 8 --runMode genomeGenerate --genomeDir <hg19_STARindex>
--genomeFastaFiles <hg19_genome.fa> --sjdbGTFfile <gencode.v19.annotation.gtf>

(2). Alignment:

.. code-block:: bash

STAR --outFilterType BySJout --runThreadN 8 --outFilterMismatchNmax 2 --genomeDir <hg19_STARindex>
--readFilesIn <un_aligned.fastq> --outFileNamePrefix (HEK293) --outSAMtype BAM
SortedByCoordinate --quantMode TranscriptomeSAM GeneCounts --outFilterMultimapNmax 1
--outFilterMatchNmin 16 --alignEndsType EndToEnd

5. **Running RiboCode to identify translated ORFs**

(1). Preparing the transcripts annotation files:

.. code-block:: bash

prepare_transcripts -g <gencode.v19.annotation.gtf> -f <hg19_genome.fa> -o <RiboCode_annot>

(2). Selecting the length range of the RPF reads and identify the P-site locations:

.. code-block:: bash

metaplots -a <RiboCode_annot> -r <HEK293Aligned.toTranscriptome.out.bam>


This step will generate a PDF file and a predefined P-site parameters file. The PDF file plots the aggregate profiles
of the distance between the 5'-end of reads and the annotated start codons or stop codons. The P-site parameters file
defines the read lengths which show strong 3-nt periodicity and the P-site locations for each length, users can modify
this file according the plots in PDF file.

(3). Detecting translated ORFs using the ribosome-profiling data:

.. code-block:: bash

RiboCode -a <RiboCode_annot> -c <config.txt> -l no -o <RiboCode_ORFs_result>


Users can use or modify the config file generated by last step to specify the information of the bam file and P-site parameters,
please refer to the example file `config.txt`_ in data folder.

**Explanation of final result files**

The *RiboCode* generates two text files as below:
The "(output file name).txt" contains the information of predicted ORFs in each
transcript; The "(output file name)_collapsed.txt" file combines the ORFs with the
same stop codon in different transcript isoforms: the one harboring the most
upstream in-frame ATG is chosen.
Some column names of the result file::

- ORF_ID: The identifier of ORFs that predicated.
- ORF_type: The type of ORF. The following ORF categories are reported:

"annotated" (overlapping annotated CDS, have the same stop with annnotated CDS)

"uORF" (in upstream of annotated CDS, not overlapping annotated CDS)

"dORF" (in downstream of annotated CDS, not overlapping annotated CDS)

"Overlap_uORF" (in upstream of annotated CDS, overlapping annotated CDS)

"Overlap_dORF" (in downstream of annotated CDS, overlapping annotated CDS"

"Internal" (in internal of annotated CDS, but in a different frame relative annotated CDS)

"novel" (in non-coding genes or non-coding transcripts of coding genes).

- ORF_tstart, ORF_tstop: the beginning and end of ORF in RNA transcript (1-based coordinate)
- ORF_gstart, ORF_gstop: the beginning and end of ORF in genome (1-based coordinate)
- pval_frame0_vs_frame1: significance levels of P-site densities of frame0 greater than of frame1
- pval_frame0_vs_frame2: significance levels of P-site densities of frame0 greater than of frame2
- pval_combined: integrated P-value

(4). (optional) Plotting the densities of P-sites for predicted ORFs

Users can plot the density of P-sites for a ORF using the "parsing_plot_orf_density" command, as example below:

.. code-block:: bash

parsing_plot_orf_density -a <RiboCode_annot> -c <config.txt> -t (transcript_id)
-s (ORF_gstart) -e (ORF_gstop)

The generated PDF plots can be edited by Adobe Illustrator.

(5). (optional) Counting the number of RPF reads aligned to ORFs

The number of reads mapping to each ORF can be obtained by the "ORF_count" command which relying on HTSeq-count package.
The first few codons and last few codons of ORF with length longer than a given value can be excluded by adjusting specific parameters.
Only the reads of a given length will be counted. For example, the reads with length between 24-36 nt aligned to
predicted ORF can be obtained by using below command:

.. code-block:: bash
ORF_count -g <RiboCode_ORFs_result.gtf> -r <ribo-seq genomic mapping file> -f 15 -l 5 -e 100 -m 26 -M 34 -o <ORF.counts>

The reads aligned to first 15 codons and last 5 codons of ORFs with length longer than 100 nt will be excluded.


Recipes (FAQ):
--------------
1. **I have a BAM/SAM file aligned to genome, how do I convert it to transcriptome-based mapping file ?**

You can use STAR aligner to generate the transcriptome-based alignment file by specifying the "--quantMode TranscriptomeSAM" parameters,
or use the "sam-xlate" command from `UNC Bioinformatics Utilities`_ .

2. **How to use multiple BAM/SAM files to identify ORFs?**

You can select the read lengths which show strong 3-nt periodicity and the corresponding P-site locations for each
BAM/SAM file, then list each file and their information in `config.txt`_ file. *RiboCode* will combine the P-site
densities at each nucleotides of these BAM/SAM files together to predict ORFs.


For any questions, please contact:
----------------------------------

Zhengtao Xiao (xzt13@mails.tsinghua.edu.cn)

Rongyao Huang (THUhry12@163.com)

Xudong Xing (xudonxing_bioinf@sina.com)

.. _SRA_Toolkit: https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software
.. _HEK293 dataset: https://trace.ncbi.nlm.nih.gov/Traces/sra/?run=SRR1630831
.. _config.txt: https://github.com/xzt41/RiboCode/blob/master/data/config.txt
.. _rRNA.fa: https://github.com/xzt41/RiboCode/blob/master/data/rRNA.fa
.. _UNC Bioinformatics Utilities: https://github.com/mozack/ubu