The authors would like to acknowledge the support from the computational resources provided by the HPCC platform of Xi&#39;an Jiaotong University. Z.X. gratefully thanks the Young Topnotch Talent Support Plan of Xi&#39;an Jiaotong University.

1. Environment setup and RiboCode installation
<ol>
	<li>Open a Linux terminal window and create a conda environment: 
	conda create -n RiboCode python=3.8</li>
	<li>Switch to the created environment and install RiboCode and dependencies: 
	conda activate RiboCode 
	conda install -c bioconda ribocode ribominer sra-tools fastx_toolkit cutadapt bowtie star samtools</li>
</ol>
2. Data preparation
<ol>
	<li>Get genome reference fil...

<ol>
	<li>Eisenberg, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+Initiation+Site+Profiling+Reveals+Widespread+Synthesis+of+Non-AUG-Initiated+Protein+Isoforms+in+Yeast.">Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.</a> Cell Systems. 11 (2), 145-160 (2020).</li><li>Spealman, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+non-AUG+uORFs+revealed+by+a+novel+regression+analysis+of+ribosome+profiling+data.">Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data.</a> Genome Research. 28 (2), 214-222 (2018).</li><li>Ingolia, N. T., Lareau, L. F., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling+of+mouse+embryonic+stem+cells+reveals+the+complexity+and+dynamics+of+mammalian+proteomes.">Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes.</a> Cell. 147 (4), 789-802 (2011).</li><li>Bazzini, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+small+ORFs+in+vertebrates+using+ribosome+footprinting+and+evolutionary+conservation.">Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation.</a> The EMBO Journal. 33 (9), 981-993 (2014).</li><li>Ingolia, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling+reveals+pervasive+translation+outside+of+annotated+protein-coding+genes.">Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.</a> Cell Reports. 8 (5), 1365-1379 (2014).</li><li>Chew, G. L., Pauli, A., Schier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+uORF+repressiveness+and+sequence+features+in+mouse,+human+and+zebrafish.">Conservation of uORF repressiveness and sequence features in mouse, human and zebrafish.</a> Nature Communications. 7, 11663 (2016).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+genome-wide+distribution+and+evolution+of+uORFs+in+eukaryotes.">Determinants of genome-wide distribution and evolution of uORFs in eukaryotes.</a> Nature Communications. 12 (1), 1076 (2021).</li><li>Guenther, U. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+helicase+Ded1p+controls+use+of+near-cognate+translation+initiation+codons+in+5'+UTRs.">The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.</a> Nature. 559 (7712), 130-134 (2018).</li><li>Goldsmith, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling+reveals+a+functional+role+for+autophagy+in+mRNA+translational+control.">Ribosome profiling reveals a functional role for autophagy in mRNA translational control.</a> Communications Biology. 3 (1), 388 (2020).</li><li>Magny, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+regulation+of+cardiac+calcium+uptake+by+peptides+encoded+in+small+open+reading+frames.">Conserved regulation of cardiac calcium uptake by peptides encoded in small open reading frames.</a> Science. 341 (6150), 1116-1120 (2013).</li><li>Stumpf, C. R., Moreno, M. V., Olshen, A. B., Taylor, B. S., Ruggero, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translational+landscape+of+the+mammalian+cell+cycle.">The translational landscape of the mammalian cell cycle.</a> Molecular Cell. 52 (4), 574-582 (2013).</li><li>Gerashchenko, M. V., Lobanov, A. V., Gladyshev, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+ribosome+profiling+reveals+complex+translational+regulation+in+response+to+oxidative+stress.">Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (43), 17394-17399 (2012).</li><li>Andreev, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+glucose+deprivation+induces+widespread+alterations+in+mRNA+translation+within+20+minutes.">Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes.</a> Genome Biology. 16, 90 (2015).</li><li>Chng, S. C., Ho, L., Tian, J., Reversade, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELABELA:+a+hormone+essential+for+heart+development+signals+via+the+apelin+receptor.">ELABELA: a hormone essential for heart development signals via the apelin receptor.</a> Developmental Cell. 27 (6), 672-680 (2013).</li><li>Pauli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toddler:+an+embryonic+signal+that+promotes+cell+movement+via+Apelin+receptors.">Toddler: an embryonic signal that promotes cell movement via Apelin receptors.</a> Science. 343 (6172), 1248636 (2014).</li><li>Stark, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+functional+elements+in+12+Drosophila+genomes+using+evolutionary+signatures.">Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures.</a> Nature. 450 (7167), 219-232 (2007).</li><li>Lin, M. F., Jungreis, I., Kellis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PhyloCSF:+a+comparative+genomics+method+to+distinguish+protein+coding+and+non-coding+regions.">PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions.</a> Bioinformatics. 27 (13), 275-282 (2011).</li><li>Slavoff, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidomic+discovery+of+short+open+reading+frame-encoded+peptides+in+human+cells.">Peptidomic discovery of short open reading frame-encoded peptides in human cells.</a> Nature Chemical Biology. 9 (1), 59-64 (2013).</li><li>Schwaid, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoproteomic+discovery+of+cysteine-containing+human+short+open+reading+frames.">Chemoproteomic discovery of cysteine-containing human short open reading frames.</a> Journal of the American Chemical Society. 135 (45), 16750-16753 (2013).</li><li>Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+annotation+and+quantitation+of+translation+by+ribosome+profiling.">Genome-wide annotation and quantitation of translation by ribosome profiling.</a> Current Protocols in Molecular Biology. , 1-19 (2013).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324 (5924), 218-223 (2009).</li><li>Xiao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+annotation+and+characterization+of+the+translatome+with+ribosome+profiling+data.">De novo annotation and characterization of the translatome with ribosome profiling data.</a> Nucleic Acids Research. 46 (10), 61 (2018).</li><li>Lin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eIF3+Associates+with+80S+Ribosomes+to+Promote+Translation+Elongation,+Mitochondrial+Homeostasis,+and+Muscle+Health.">eIF3 Associates with 80S Ribosomes to Promote Translation Elongation, Mitochondrial Homeostasis, and Muscle Health.</a> Molecular Cell. 79 (4), 575-587 (2020).</li><li>. AGAT: Another Gff Analysis Toolkit to handle annotations in any GTF/GFF format Available from: <a target="_blank" href="https://agat.readthedocs.io/en/latest/gff_to_gtf.html">https://agat.readthedocs.io/en/latest/gff_to_gtf.html</a> (2020)</li><li>. Gene Expression Omnibus Available from: <a target="_blank" href="https://www.ncbi.nim.nih.gov/geo">https://www.ncbi.nim.nih.gov/geo</a> (2002)</li><li>Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+profiling+strategy+for+monitoring+translation+in+vivo+by+deep+sequencing+of+ribosome-protected+mRNA+fragments.">The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.</a> Nature Protocols. 7 (8), 1534-1550 (2012).</li><li>. STAR manual Available from: <a target="_blank" href="https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf">https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf</a> (2022)</li><li>. The genetic codes Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi">https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi</a> (2019)</li><li>. RiboMiner Available from: <a target="_blank" href="https://github.com/xryanglab/RiboMiner">https://github.com/xryanglab/RiboMiner</a> (2020)</li><li>Ingolia, N. T., Hussmann, J. A., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling:+global+views+of+translation.">Ribosome profiling: global views of translation.</a> Cold Spring Harbor Perspectives in Biology. 11 (5), 032698 (2018).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+mapping+of+translation+initiation+sites+in+mammalian+cells+at+single-nucleotide+resolution.">Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (37), 2424-2432 (2012).</li><li>Gao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+profiling+of+initiating+ribosomes+in+vivo.">Quantitative profiling of initiating ribosomes in vivo.</a> Nature Methods. 12 (2), 147-153 (2015).</li><li>Spealman, P., Naik, A., McManus, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=uORF-seqr:+A+Machine+Learning-Based+approach+to+the+identification+of+upstream+open+reading+frames+in+yeast.">uORF-seqr: A Machine Learning-Based approach to the identification of upstream open reading frames in yeast.</a> Methods in Molecular Biol. 2252, 313-329 (2021).</li><li>. RiboCode Available from: <a target="_blank" href="https://github.com/xryanglab/RiboCode">https://github.com/xryanglab/RiboCode</a> (2018)</li><li>Sharma, P., Wu, J., Nilges, B. S., Leidel, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humans+and+other+commonly+used+model+organisms+are+resistant+to+cycloheximide-mediated+biases+in+ribosome+profiling+experiments.">Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments.</a> Nature Communications. 12 (1), 5094 (2021).</li></ol>

Ribosome profiling offers an unprecedented opportunity to study the ribosomes' action in cells at a genome scale. Precisely deciphering the information carried by the ribosome profiling data could provide insight into which regions of genes or transcripts are actively translating. This step-by-step protocol provides guidance on how to use RiboCode to analyze ribosome profiling data in detail, including package installation, data preparation, command execution, result explanation, and data visualization. The analysis resu...

Recently, a growing body of studies has revealed widespread production of peptides translated from ORFs of coding genes and the previously annotated genes as noncoding, such as long noncoding RNAs (lncRNAs)1,2,3,4,5,6,7,8. These translated ORFs are regulated or induced by cells to respond to environmental changes, stress, and cell differentiation1,8,9,10,11,12,13. The translation products of some ORFs have been demonstrated to play important regulatory roles in diverse biological processes in development and physiology. For example, Chng et al.14 discovered a peptide hormone named Elabela (Ela, also known as Apela/Ende/Toddler), which is critical for cardiovascular development. Pauli et al. suggested that Ela also acts as a mitogen that promotes cell migration in the early fish embryo15. Magny et al. reported two micropeptides of less than 30 amino acids regulating calcium transport and affecting regular muscle contraction in the Drosophila heart10.
It remains unclear how many such peptides are encoded by the genome and whether they are biologically relevant. Therefore, systematic identification of these potentially coding ORFs is highly desirable. However, directly determining the products of these ORFs (i.e., protein or peptide) using traditional approaches such as evolutionary conservation16,17 and mass spectrometry18,19 is challenging because the detection efficiency of both approaches is dependent on the length, abundance, and amino acid composition of the produced proteins or peptides. The advent of ribosome profiling, a technique for identifying the ribosome occupancy on mRNAs at nucleotide resolution, has provided a precise way to evaluate the coding potential of different transcripts3,20,21, irrespective of their length and composition. An important and frequently used feature for identifying actively translating ORFs using ribosome profiling is the three-nucleotide (3-nt) periodicity of the ribosome&#39;s footprints on mRNA from the start codon to the stop codon. However, ribosome profiling data often have several issues, including low and sparse sequencing reads along ORFs, high sequencing noise, and ribosomal RNA (rRNA) contaminations. Thus, the distorted and ambiguous signals generated by such data weaken the 3-nt periodicity patterns of ribosomes&#39; footprints on mRNA, which ultimately makes the identification of the high-confidence translated ORFs difficult.
A package named &#34;RiboCode&#34; adapted a modified Wilcoxon-signed-rank test and P-value integration strategy to examine whether the ORF has significantly more in-frame ribosome-protected fragments (RPFs) than off-frame RPFs22. It was demonstrated to be highly efficient, sensitive, and accurate for de novo annotation of the translatome in simulated and real ribosome profiling data. Here, we describe how to use this tool to detect the potential translating ORFs from the raw ribosome profiling sequencing datasets generated by the previous study23. These datasets had been used to explore the function of EIF3 subunit &#34;E&#34; (EIF3E) in translation by comparing the ribosome occupancy profiles of MCF-10A cells transfected with control (si-Ctrl) and EIF3E (si-eIF3e) small-interfering RNAs (siRNAs). By applying RiboCode to these example datasets, we detected 5,633 novel ORFs potentially encoding small peptides or proteins. These ORFs were categorized into various types based on their locations relative to the coding regions, including upstream ORFs (uORFs), downstream ORFs (dORFs), overlapped ORFs, ORFs from novel protein-coding genes (novel PCGs), and ORFs from novel nonprotein-coding genes (novel NonPCGs). The RPF read densities on uORFs were significantly increased in EIF3E-deficient cells compared to control cells, which might be at least partially caused by the enrichment of actively translating ribosomes. The localized ribosome accumulation in the region from the 25th to 75th codon of EIF3E-deficient cells indicated a blockage of translation elongation in the early stage. This protocol also shows how to visualize the RPF density of the desired region for examining the 3-nt periodicity patterns of ribosome footprints on identified ORFs. These analyses demonstrate the powerful role of RiboCode in identifying translating ORFs and studying the regulation of translation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A computer/server running Linux</td>
			<td>Any</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Anaconda or Miniconda</td>
			<td>Anaconda</td>
			<td>-</td>
			<td>Anaconda: https://www.anaconda.com; Miniconda:https://docs.conda.io/en/latest/miniconda.html</td>
		</tr>
		<tr>
			<td>R</td>
			<td>R Foundation</td>
			<td>-</td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td>Rstudio</td>
			<td>-</td>
			<td>https://www.rstudio.com/</td>
		</tr>
	</tbody>
</table>

de novo identification of actively translated open reading frames with ribosome profiling data

Identification of open reading frames (ORFs), especially those encoding small peptides and being actively translated under specific physiological contexts, is critical for&#160;comprehensive annotations of context-dependent translatomes. Ribosome profiling, a technique for detecting the binding locations and densities of translating ribosomes on RNA, offers an avenue to rapidly discover where translation is occurring at the genome-wide scale. However, it is not a trivial task in bioinformatics to efficiently and comprehensively identify the translating ORFs for ribosome profiling. Described here is an easy-to-use package, named RiboCode, designed to search for actively translating ORFs of any size from distorted and ambiguous signals in ribosome profiling data. Taking our previously published dataset as an example, this article provides step-by-step instructions for the entire RiboCode pipeline, from preprocessing of the raw data to interpretation of the final output result files. Furthermore, for evaluating the translation rates of the annotated ORFs, procedures for visualization and quantification of ribosome densities on each ORF are also described in detail. In summary, the present article is a useful and timely instruction for the research fields related to translation, small ORFs, and peptides.

The example ribosome profiling datasets were deposited in the GEO database under the accession number GSE131074. All the files and codes used in this protocol are available from Supplemental files 1-4. By applying RiboCode to a set of published ribosome profiling datasets23, we identified the novel ORFs actively translated in MCF-10A cells treated with control and EIF3E siRNAs. To select the RPF reads that are most likely bound by the tra...

Watch this Scientific Journal Video about De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data at JoVE.com

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Translating ribosomes decode three nucleotides per codon into peptides. Their movement along mRNA, captured by ribosome profiling, produces the footprints exhibiting characteristic triplet periodicity. This protocol describes how to use RiboCode to decipher this prominent feature from ribosome profiling data to identify actively translated open reading frames at the whole-transcriptome level.

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Identification of open reading frames (ORFs), especially those encoding small peptides and being actively translated under specific physiological ...

Interpretation of the sequencing data generated by the ribosome profiling experiment is critical for quantitatively measuring the translational activities of ribosomes on mRNA, and for studying the mechanisms of translational regulation. In this protocol, we will describe the computational procedure for utilizing the ribosome profiling data and RiboCode, a command line tool to decode mRNA translation at the genome-wide scale and single nucleotide resolution. This method allows to search for the novel peptides arising from the genomic regions outside of the annotated protein coding genes, and offers an opportunity to quantify the rate of mRNA translation.To begin, open a Linux terminal window, and create a conda environment by executing the command. Switch to the created environment, and install RiboCode and dependencies by executing the command. To get the genome reference files for the reference sequence, go to the Ensembl website, then click download, followed by FTP download.Click the FASTA option in the column DNA FASTA, and select the row where species is human, shown in the table on the website page. On the Ensembl website page, copy the link as mentioned in the text, then download and unzip the files in the terminal by executing the command. For reference annotation, right click GTF in the column gene sets in the last open webpage.Copy the link, and download it using the command. To get rRNA sequences, open UCSC genome browser, then click tools, and select table browser in the dropdown list. On the UCSC genome browser page, specify mammal for clade, human for genome, all tables for group, R mask for table, and genome for region.For filter, click create to go to a new page, and set rep class as does match rRNA. Click submit, and then set the output format to sequence, and output file name as HG38_rRNA. FA.Finally, click get output, and then select get sequence to retrieve the rRNA sequence.To get ribosome profiling datasets from sequence read archive, download the replicate samples of the si-eIFe treatment group, and rename them by executing the command. Then download the replicate samples of the control group, and rename them by executing the command. To remove rRNA contamination, start indexing rRNA reference sequences by executing the command.After indexing, align the reads to rRNA reference to rule out the reads originating from rRNA by executing the command. Start by creating a genome index by executing the command. Then align the clean reads with no rRNA contamination to the created reference by executing the command, and then sort and index alignment files by executing the command.Prepare the transcript annotations by executing the command. Select ribosome protected fragments of specific lengths, and identify their P-site positions by executing the command. Edit the configuration files for each sample and merge them.Then run RiboCode by executing the command. The frequency distribution of the lengths of the reads showed that most ribosome protected fragments correspond to 25 to 35 nucleotides. The P-site locations for different lengths of ribosome protected fragments were determined by examining the distances from their five prime ends to the annotated start and stop codons.The mapping results show that 10, 394 genes encode for annotated open reading frames. Further, 509 and 168 genes encode for upstream and downstream open reading frames, while 939 genes encode for either upstream or downstream open reading frames, overlapped with known annotated open reading frames. Further, 68 protein coding genes and 2, 601 non-coding genes encode for novel open reading frames.Length distribution showed that upstream, downstream, novel, and overlapped open reading frames were shorter than the annotated open reading frames. Relative ribosome protected fragment counts were calculated for each open reading frame, revealing that the ribosome densities of upstream open reading frames were significantly higher in eIF3e deficient cells than in control cells. The metagene analysis revealed that a mass of ribosomes stalled between codons 25 and 75 downstream of the start codon, suggesting that the translation elongation might be blocked early in eIF3e deficient cells.The P-sites density profiles for upstream open reading frames of PSMA6 and downstream open reading frames of gene SENP3-EIF4A1 were examined, demonstrating the periodicity patterns and densities of ribosome protected fragments. Checking the locations of reads around the start and stop codons of known protein coding regions is necessary for evaluating the periodic properties of reads for each length. RiboCode, together with another command line tool, RiboMiner can also perform quality control and multiple analyses such as quantifying and visualizing the ribosomes'occupancies on the predicted open reading frames.This computational tool provides a high throughput way to identify uncanonical translation events with ribosome profiling data in specific physiological contexts, and how the translation modulates in response to the stimulus.

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3.3K 조회수.  Xi’an Jiaotong University Health Science Center. 리보솜 프로파일링 실험에 의해 생성된 시퀀싱 데이터의 해석은 mRNA에서 리보솜의 번역 활동을 정량적으로 측정하고 번역 조절의 메커니즘을 연구하는 데 매우 중요합니다. 이 프로토콜에서는 리보솜 프로파일링 데이터를 활용하기 위한 계산 절차와 게놈 전체 규모 및 단일 뉴클레오티드 분해능에서 mRNA 번역을 디코딩하는 커맨드 라인 도구인 RiboCode를 설명합니다. 이 방법?...