Rationale for sending manned mission to another star? You can add Python and the pip directory to the PATH in a bit. Subread-featureCounts-limma/voom pipeline has been found to be one of the best-performing pipelines for the analyses of RNA-seq data by the SEquencing Quality Control (SEQC) study, the third stage of the well-known MicroArray Quality Control (MAQC) project [8]. hierarchical), GitHub integration to facilitate contributing to EasyBuild, EasyBuild at Jlich Supercomputing Centre. 464 Bearsden Rd directory for successful installation, into the easybuild subdirectory. Note that we are using -fcommon Counting RNA-seq reads is complex because of the need to accommodate exon splicing. Connect and share knowledge within a single location that is structured and easy to search. Gene annotations in GTF format. has occurred, and the installation will be interrupted. If not, don't worry. That's not where our toolchain compiler is installed, exercise, but it may help with the step-wise approach we'll take and When you run featureCounts in paired-end mode (using the -p argument) it counts fragments rather than reads, as stated in the documentation: If specified, fragments (or templates) will be counted instead of reads. shell command in the error message, which is a simple way to try include ran correctly, and it will continue with the rest of the installation procedure. Can I get help on an issue where unexpected/illegible characters render in Safari on some HTML pages? Browse other questions tagged, Where developers & technologists share private knowledge with coworkers, Reach developers & technologists worldwide, how to fix featureCounts in miniconda (Linux) with error "featureCounts: invalid option -- 'r'", Building a safer community: Announcing our new Code of Conduct, Balancing a PhD program with a startup career (Ep. 4.Besides UNIX version, it also has R-based version. Can you spot something suspicious here? It has been widely used in CHIP-Seq and RNA-Seq. But the second one featureCounts -a GCF_000837865.1_ViralProj14074_genomic.gtf -g gbkey -o SRR11074360_1_10k.sorted_biotype.featureCounts.txt -p -s 2 SRR11074360_1_10k.sorted.bam fails with the error: ERROR: no features were loaded in format GTF. I'm having a very strange error when running featureCounts on a tiny test file: it says it's generating counts in the summary.txt file, but the 7th column of the counttable file is empty: From your first cat | awk | etc. See for example this output line from our earlier example error message: You can open this file with your favorite text editor or a tool like less The featureCounts program in this version will report the details of the first read pair that was found not properly paired. When the code is run this way, no reads are reported in the output file (although in theory they should, if you look at the log!/, If, however, I paste the flag from the command line (i.e. This is because you did pair-end read assignment. In that case, you will have the dive into the log file that is created by EasyBuild for I have surprisingly low counts when running featureCounts on some (single-end) RNA-seq data mapped on C. elegans genome using hisat2.. To more easily show the problem, I generated a small subset of the bam file and of the annotation file I'm using. Its worth to mention that the citation of htseq-count is favourable, lots of users are still use htseq. We have released a patched version of Subread package (1.4.6-p3). The comment above the buildopts definition makes it clear that the -fcommon The dUTP protocol is commonly used for performing strand-specific sequencing. Hence featureCounts found no features for read-pair 7040_1110_13610641. Policy. Hence, in summary, the controversial between two tools could be ignored in real data analysis. You may try to set '-s 1', or there may be an issue with the sample prep for these samples. For example, if two genes were found to both overlap with a reads pair fragment but one gene was found to overlap with only one read and the other with both reads from that fragment, featureCounts will assign that fragment to the gene overlapping with both reads. Please let me know if additional information is required. log for HDF5): It can be useful to look for the first error that occurred in the output of a command, since subsequent errors are Here is an example of an EasyBuild error message (slightly reformatted for clarity): Let's break this down a bit: during the build step of the installation When I try to map to gene_name featureCounts says that gene_name is not found in the 9th column of the .gtf file. to the most recently updated build log. Did you try sorting by name and see if it works? Does Russia stamp passports of foreign tourists while entering or exiting Russia? Possible values include: 0 (unstranded), 1 (stranded) and 2 (reversely stranded). privacy statement. Note that we are using -fcommon Can you fix the next problem you run into? Because I filtered out the unmapped sequences in advance, the overall assignment ratio displayed by Hisat2 was 100%, and the multi-mapping ratio was only 0.3%. rather than error. What's wrong now? Yes, I'm in bash, but, unfortunately, that's not the case. If you continue to use this site we will assume that you are happy with it. When EasyBuild detects that something went wrong, it will usually produce a 05-17-2015, 06:01 PM. OK fine, but now what? The following is the Hisat2 command. In some cases there won't be any useful information in there however, I guess I didn't know much about web formats. Ideally, you should use something like snakemake, not a raw python script. How to say They came, they saw, they conquered in Latin? Therefore FC rightly declares that it cannot find any "features". Asking for help, clarification, or responding to other answers. By clicking Accept all cookies, you agree Stack Exchange can store cookies on your device and disclose information in accordance with our Cookie Policy. the easyconfig file can be downloaded from: This way, EasyBuild will download the source file when running eb subread.eb. and you'll miss others that do include errors but mention ERROR or Error informative messages produced by both the EasyBuild framework and the easyblock Also make sure that the pre-installed software stack is available, when you have Vim mapped to always print two? How does a government that uses undead labor avoid perverse incentives? the read with flag 147) was mapped to the negative strand, featureCounts flipped the strandness of R2, suggesting that the whole fragment was from the positive strand of the chromosome. Sorry for the wrong description in the previous post. We have released a patched version of Subread package (1.4.6-p3). Regarding the example read you showed, how did you obtain its mapping location? in /easybuild/sources/s/Subread. There is no significant improvement with this setting of '-s 1'. that's somewhere under /easybuild/software. Strictly speaking the configuration doesn't matter much for the sake of this When an installation fails the corresponding build directory is not cleaned up Does the conduit for a wall oven need to be pulled inside the cabinet? Also included in this software suite is a very e cient SNP caller { ExactSNP. But which one is better? Why is Bb8 better than Bc7 in this position? Policy. I have had the best luck using the command line version. How to say They came, they saw, they conquered in Latin? that are not actually errors at all (like the compilation of an error.c file), I had a similar problem with --fraction which led me here! 1. a data matrix containing read counts for each feature or meta-feature for each library. The package you are looking for can therefore not be installed on windows using conda. Does the policy change for AI-generated content affect users who (want to) Is there a reason beyond protection from potential corruption to restrict a minister's ability to personally relieve and appoint civil servants? Is there any difference between calling featureCounts in command prompt (or Terminal on Mac) and calling it in Linux/miniconda3? The other read-pair in your screenshot, 7040_1110_9490926, has R1 mapped to the negative strand (flag = 83) and R2 mapped to the positive strand of the chromosome (flag = 163). Lastly, regarding some of your samples having only 2-3% of reads assigned, I am not sure what is the reason. Hence featureCounts takes it as the whole fragment of 7040_1110_9490926 was from the negative strand of the chromosome. If you look at the full output in the log file you can see that the correct option to check the version of the featureCounts command is "-v" rather than "--version", so we need to fix this in the easyconfig file. email: [email protected]. featureCounts is a general-purpose read summarization function that can assign mapped reads from genomic DNA and RNA sequencing to genomic features or meta-features. every installation, which is located in the unique temporary directory for the EasyBuild session. In this pipeline, the vitalstep is to estimate the reads count of each genomic features. since the actual error message(s) could only appear way later, perhaps even after Hello I'm trying to use featureCounts for multiple files using python script. This is a fictitious example of course, but hopefully it gives you a feeling Markdown formatting rules are that 2 new line characters move to a new paragraph in the rendered markdown (like an HTML
tag) while one new line just moves the cursor in the raw markdown text (again, like in HTML). environment carefully. "featureCounts preforms precise read assignment by comparing mapping location of every base in the read or fragment with the genomic region spanned by each feature" (paper) | Screenshot: Ashley Gelwix. DESCRIPTION Version 2.0.1 ## Mandatory arguments: -a <string> Name of an annotation file. with the full output of the command that failed. The installation fails because the source file subread-2.0.1-source.tar.gz In my opinion, featureCounts can make a clear distinction for those features that overlap with different numbers of reads from the same fragment, meanwhile I think htseq-count is too conservative and get lower counts. Wait a minute Why is make using /usr/bin/g++ for the compilation?! the easyconfig file? The function takes as input a set of SAM or BAM files containing read mapping results. Is it possible to type a single quote/paren/etc. exercise, but it may help with the step-wise approach we'll take and Sign in I have a similar problem both for sorted and unsorted output from star. GTF/GFF format by default. In any case, try providing the full path to featureCounts in your system(). as simple as eating a piece of cake but not cheese one! I'm new to conda and trying to install featureCounts contained in DESeq2 package. If you look at the results of my head command, I'm getting exactly what I would expect: And all of the rest of the lines of the file (wc -l identifies 47071 lines in the file, with each line looking like line 3) have nothing (not even a zero!) and focus on a couple of EasyBuild aspects that can be helpful in that context, To do this, we're going to use the featurecounts program from the subread package in R. This package can be installed either as an R package or as a command-line program. OK, so I figured it out, and it was not very intuitive to debug! Site design / logo 2023 Stack Exchange Inc; user contributions licensed under CC BY-SA. When EasyBuild detects that something went wrong, it will usually produce a What's wrong now? Oh my that's pretty ancient (GCC 4.8.5 was released in June 2015). Input files: The input of featureCounts could be SAF files rather than GTF/GFF files. DESeq, DESeq2) across RNA-Seq analysis. Don't give up now, try one last time and fix the last problem that occurs Now the installation itself works but the sanity check fails, grasping the solutions. I used Hisat2 to assign paired-end strand-specific transcriptomic sequences (rRNA removed) to a reference genome. to the start of the output for a command using "INFO running cmd" as a search pattern, and then looking for patterns This allows you to dive in and check for clues in the files that are stored there. the error is pretty obvious: The easyconfig file hard specifies the -fast compiler flag via the CFLAGS argument to the build command: EasyBuild sets up the build environment, so there should be no need that are not actually errors at all (like the compilation of an error.c file), I am analysing data in C. elegans using featureCounts and am having problems mapping to gene_name rather than gene_id. Enabling a user to revert a hacked change in their email, QGIS - how to copy only some columns from attribute table. To learn more, see our tips on writing great answers. (missing mate or the mate is not the next read)'. Can you fix the next problem you run into? For convenience, we picked an unassigned sequence, and named it unassigned.fa, which was 150 nt. However, htseq-count will take this fragment as ambiguous and will not assign it to any gene. An easy way to fix this problem is to replace the -fast with -Ofast, 7040_1110_9490926 was assigned to features 1_2, while 7040_1110_13610641 was reported as "no features" ) was observed in the result of featureCounts run with -s 2. FeatureCounts works fine mapping to gene_id with the following code: I can see gene_name in the gtf file when I do head .gtf so I believe it should be possible to map to gene_name rather than gene_id (although maybe I'm mistaken!). It can be used to count both RNA-seq and genomic DNA-seq reads. counts_junction (optional) a data frame including the number of supporting reads for each exon-exon junction, genes that junctions belong to, chromosomal coordinates of splice sites, etc. CMakeOutput.log or CMakeError.log for clues, which are sneakily hidden by CMake in a CMakeFiles subdirectory of the build directory. and focus on a couple of EasyBuild aspects that can be helpful in that context, Command-line tool R. featureCounts is a very efficient read quantifier. featureCounts -a GCF_000837865.1_ViralProj14074_genomic.gtf -g gene_id -t gene -o SRR11074360_1_10k.sorted_gene.featureCounts.txt --extraAttributes gene_name -p -s 2 SRR11074360_1_10k.sorted.bam. that's somewhere under /easybuild/software. like "error:" from there. Cartoon series about a world-saving agent, who is an Indiana Jones and James Bond mixture. At ENA, the sequencing reads are directly available in FASTQ or SRA formats, which will be explained below. and that the EasyBuild module is loaded (unless you installed EasyBuild Your tags are already good but I've added an Rsubread tag, which will make absolutely sure that the Rsubread developers see your post. and inspect the config.log file in the build directory to determine the underlying cause of an error. to be build command to replace the -fast with $CFLAGS, ran correctly, and it will continue with the rest of the installation procedure. Login before adding your answer. I have downloaded the following .gtf file from WormBase https://downloads.wormbase.org/releases/WS280/species/c_elegans/PRJNA13758/c_elegans.PRJNA13758.WS280.canonical_geneset.gtf.gz. Please check the following information of bam file and gff file. via the featureCounts command, which should look like this: "High performance read alignment, quantification and mutation discovery", # download from https://download.sourceforge.net/subread/subread-2.0.1-source.tar.gz, 'd808eb5b1823c572cb45a97c95a3c5acb3d8e29aa47ec74e3ca1eb345787c17b'. using -fno-common by default. For the bioinformatian who liked to perform RNA-Seq or CHIP-Seq analysis in Windows/MacOS, featureCounts is the best choice. If this is mouse data, then from my experiences the percentage of assigned reads is typically around 50-70%, so your percentage is within the range. In this part we take a look at how you can troubleshoot a failing installation, Check your work by manually loading the module and checking the version via the featureCounts command, which should look like this: $ featureCounts-v featureCounts v2.0.1 next: Module naming schemes - (back to overview page) The metatranscriptomes were from mixed uncultured microbiota. The source tarball is fairly large (23MB), so don't be alarmed if the download takes a little while. The high computational efficiency of featureCounts is due to its ultrafast feature search algorithm and its highly efficient implementation entirely using the C programming language. Noise cancels but variance sums - contradiction? The low successful assignment ratio of FeatureCounts, Traffic: 239 users visited in the last hour, User Agreement and Privacy For example: The eb command supports a handy little option that prints the location to take a look at the information collected in the log file, which includes Because the "R2" read in 7040_1110_13610641 (i.e. So far a couple of software(e.g. To subscribe to this RSS feed, copy and paste this URL into your RSS reader. In Germany, does an academic position after PhD have an age limit? 576), AI/ML Tool examples part 3 - Title-Drafting Assistant, We are graduating the updated button styling for vote arrows. Traffic: 239 users visited in the last hour, featureCounts will just count the data as unstranded, User Agreement and Privacy describing how the installation is progressing; how the build environment was set up: which modules were loaded, which environment variables were set; the exact shell commands that were executed, and in which directory they were run; the full output produced by these commands, and their exit code. EasyBuild truncating the command output (only the 300 first characters of the output are shown): If you open the log file and scroll to the end, and you will miss others that do include errors but mention ERROR or Error Does anyone know what does the error message "invalid option --'r'" mean? Thanks for giving very detailed read mapping results and counting results. either by scrolling or by searching for specific patterns. of suggestions of patterns you can use to locate errors: Using "error" as a search pattern is not very useful: you will hit a lot of log lines environment carefully. The 83 flag represents that read paired (0x1), read mapped in proper pair (0x2), read reverse strand (0x10) and first in pair (0x40). To observe it, lets firstly check how htseq-count do the counting the figure is taken from htseq manual. informative messages produced by both the EasyBuild framework and the easyblock Try to install the subread.eb easyconfig file, see what happens. use $HOME/easybuild as prefix, and to use /tmp/$USER as buildpath: Check your configuration via eb --show-config. open the build log of the last failed EasyBuild session in an editor: Usually you want to go to the end of the log file and then work your way up, EasyBuild truncating the command output (only the 300 first characters of the output are shown): If you open the log file and scroll to the end, 6th Annual training course on Viral Bioinformatics and Genomics (21 25 August 2023), 4th Annual Training Course on Viral Bioinformatics and Genomics (20-24th Aug, 2018), Extensive but not comprehensive compilation of de-novo assemblers, A compilation of conversion tools for BED, SAM/BAM, psl, pslx, blast tabular and blast xml, 3rd Viral Bioinformatics and Genomics Training Course (7th 11th August 2017), Extraction of FASTA sequences from Oxford Nanopore fast5 files a comparison of tools, 2nd Viral Bioinformatics and Genomics Training Course (1st 5th August 2016). Why do some images depict the same constellations differently? as an escape mechanism here: it would be better to fix the source code Maybe this argument is not correctly given? Not the answer you're looking for? It can be used to summarize RNA-seq reads and gDNA-seq reads to a variety of genomic features such as genes, exons, promoters, gene bodies and genomic bins. We want to encourage our community members to share their knowledge and help each other out by answering questions related to sequencing technologies, genomics, and bioinformatics. It is included in the Bioconductor Rsubread package and also in the SourceForge Subread package. by pasting from running featureCounts without arguments) all is well and I get. Default value is 0 (ie. from scratch, you can use the --module-only option to only run the should be used as toolchain: We don't have this GCC version installed, but we do have GCC 10.2.0: Edit the easyconfig file so it contains this: With the first two problems fixed, now we can actually try to build the software. Thank you very much for your detailed and patient answer! Thanks! Then I wanted to use FeatureCounts to get read counts based on the bam file obtained by Hisat2, below is my command, but successful assignment ratio was only about 54%, the attribute of others was "Unassigned_NoFeatures". Is there a way to map to gene_name rather than gene_id? you will need to install it another way, maybe by downloading from their website directly. You may need to talk to your sequencing facility to see if the samples of concern were indeed generated from strand-specific sequencing. using the easyconfig file that is provided below. The low successful assignment ratio of FeatureCounts. to hard specify compiler flags (certainly not incorrect ones). You can do this by first navigating P.S How do I create a new line in this thing? To subscribe to this RSS feed, copy and paste this URL into your RSS reader. A single integer value (applied to all input files) or a string of comma-separated values (applied to each corresponding input file) should be provided. failed (exit code 2, so not zero). 576), AI/ML Tool examples part 3 - Title-Drafting Assistant, We are graduating the updated button styling for vote arrows. Counting occurrence of specific character per gene in fasta file using python, How to count sequences in a fasta file using Bioperl, scikit-bio extract genomic features from gff3 file, Counting the number of paralogues for mouse genes gives me the wrong frequency, how to match gene probe ID with gene symbol in dataframe in R, Where developers & technologists share private knowledge with coworkers, Reach developers & technologists worldwide, For others looking you can use this online tool, FeatureCounts - correct GTF file for matching to gene name (not gene_id), https://downloads.wormbase.org/releases/WS280/species/c_elegans/PRJNA13758/c_elegans.PRJNA13758.WS280.canonical_geneset.gtf.gz, Building a safer community: Announcing our new Code of Conduct, Balancing a PhD program with a startup career (Ep. procedure EasyBuild was running make as a shell command, which Even if I 'enter' it just stays in a paragraph. To learn more, see our tips on writing great answers. Inbuilt annotations (SAF format) is available in 'annotation' directory of the package. CMakeOutput.log or CMakeError.log for clues, which are sneakily hidden by CMake in a CMakeFiles subdirectory of the build directory. Example run like this. It seems that you mistyped option name, it should be --readExtension3 with two dashes in front of it and without 's' in the end. I've added four channels: r, conda-forge, defaults, and bioconda. things like: Note that the installation log is also copied into each software installation FeatureCounts works fine mapping to gene_id with the following code: . automatically, that is only done for successful installations. In this movie I see a strange cable for terminal connection, what kind of connection is this? Thanks for contributing an answer to Stack Overflow! In addition to the BAM files, we also need to provide featureCounts with an annotation file. the command was already running for several minutes. Oh my that's pretty ancient (GCC 4.8.5 was released in June 2015). The 99 flag represents that read paired (0x1), read mapped in proper pair (0x2), mate reverse strand (0x20) and first in pair (0x40). the error is pretty obvious: The easyconfig file hard specifies the -fast compiler flag via the CFLAGS argument to the build command: EasyBuild sets up the build environment, so there should be no need By clicking Accept all cookies, you agree Stack Exchange can store cookies on your device and disclose information in accordance with our Cookie Policy. a checksum error on a downloaded source or patch file; required dependencies that are not specified in the easyconfig file; running out of available memory or disk space; a segmentation fault caused by a flipped bit triggered by a cosmic ray (. Would sending audio fragments over a phone call be considered a form of cryptology? is not found: In this case, the problem is that the easyconfig file does not specify Try to install the subread.eb easyconfig file, see what happens. yourself): For this exercise, make sure EasyBuild is configured to to ensure that $CFLAGS will be expanded to its value when the build command is run. Would it be possible to build a powerless holographic projector? This Python based software is developed by the scientistsfrom European Molecular Biology Laboratory, who also took part in the development of many well -knowndifferential expression tools (e.g. shell command in the error message, which is a simple way to try include When using less to view a log file, you can navigate it by: It can also be helpful to zoom in on a specific step of the installation procedure, Next, check here for . Find centralized, trusted content and collaborate around the technologies you use most. Making statements based on opinion; back them up with references or personal experience. In Germany, does an academic position after PhD have an age limit? I dont know! often fallout from earlier errors. to ensure that $CFLAGS will be expanded to its value when the build command is run. The common approach is to summarize counts at the gene level, by counting all reads that overlap any exon for each gene. available for the installation and the build directory where most software is Glasgow Can I also say: 'ich tut mir leid' instead of 'es tut mir leid'? Asking for help, clarification, or responding to other answers. So far there are two major feature counting tools: featureCounts (Liao et al.) For paired-end data, a fragment (or template) is said to overlap a feature if any of the two reads from that fragment is found to overlap the feature" (webpage) and. In this method, gene annotation file from RefSeq or Ensembl is often used for this purpose. According to the mapping locations of the paired reads 7040_1110_13610641, the paired reads 7040_1110_13610641 should be assigned to feature 1-3, but it was labeled with Unassigned_NoFeatures. So, for featureCounts, there should be a relatively high assignment ratio. I'm trying to run pipeline with a BK virus GTF file (contents of the GTF file are here) which does not contain exon as a count-type. Thanks for contributing an answer to Stack Overflow! hierarchical), GitHub integration to facilitate contributing to EasyBuild, EasyBuild at Jlich Supercomputing Centre. Developments in sequencing technologies and methodologies have transformed the field of epigenetics, giving researchers a better way to understand the complex world of gene regulation and heritable modifications. Here is an example of an EasyBuild error message (slightly reformatted for clarity): Let's break this down a bit: during the build step of the installation Or is it easier to convert to gene_name after featureCounts? to your account. the easyconfig file can be downloaded from: This way, EasyBuild will download the source file when running eb subread.eb. 4.Besides UNIX version, it also has R-based version. Your next step in this case should probably be figuring Can you fix the problem you run into, perhaps without even changing The text was updated successfully, but these errors were encountered: You signed in with another tab or window. Single-NPN driver for an N-channel MOSFET. Mahmoud classifies variant detection work into two main groups: short variants (<50, https://www.seqanswers.com/forum/sitwledge-and-win, A Review of Next-Generation Sequencing Methods for Studying EpigeneticsPart 1, Differential Expression and Data Visualization: Recommended Tools for Next-Level Sequencing Analysis, Variant Analysis and Genome Assembly: Recommended Tools for Next-Level Sequencing Analysis, Genetic Discoveries Transform Our Understanding of Primate Diversity and Behavior, Deep Sequencing Unearths Novel Genetic Variants: Enhancing Precision Medicine for Vascular Anomalies, Unveiling Genetic Associations Through Transcription Factor Binding Quantitative Trait Loci, Exploring French-Canadian Ancestry: Insights into Migration, Settlement Patterns, and Genetic Structure. automatically, that is only done for successful installations. In the case of RNA-Seq, the features are typically genes, where each gene is considered here as the union of all its exons. so you can gradually fix the problem you'll encounter. Making statements based on opinion; back them up with references or personal experience. R2) to its opposite strand. I'm sure I understand, why featureCounts perceives the input bam file as being NOT sorted, when I specifically chose to output a SORTED bam file from my star run? By clicking Post Your Answer, you agree to our terms of service and acknowledge that you have read and understand our privacy policy and code of conduct. like the error messages produced by EasyBuild, the detailed log file that is :) ruby; bash; terminal; osx-mountain-lion; homebrew; Share. Does the policy change for AI-generated content affect users who (want to) `conda skeleton` command produces invalid choice error, conda, "Solving environment: failed" error on Linux CentOS 7, Conda gives error when doing "source activate" in cmder, rstudio-server error: /bin/sh: x86_64-conda_cos6-linux-gnu-cc: command not found, `R` not working in `anaconda 3` on `Mojave`, Install new environment miniconda, unrecognized arguments:.yml, UnsatisfiableError while installing Miniconda, Miniconda: issue with shell configuration/initialization. This option is only applicable for paired-end reads; single-end reads are always counted as reads. -o <string> Name of output file including read counts. being compiled before it actually gets installed. strange results with featureCounts. failed (exit code 2, so not zero). Remember though: no peeking before you tried to solve each step yourself! Is "different coloured socks" not correct? Then, I added "-R" to see the unassigned transcriptomic sequences. is "-v" rather than "--version", so we need to fix this in the easyconfig file. has occurred, and the installation will be interrupted. Meta-features used for read counting will be extracted from annotation using the provided value. This should be a twocolumn comma-delimited text file. 5.featureCounts is more liberal than htseq-count, it could get more counts especially for pair-ended reads. So, should I use unstranded read counting method to re-generate count files, even if the libraries were constructed by dUTP method, for example the following command. Site design / logo 2023 Stack Exchange Inc; user contributions licensed under CC BY-SA. featureCounts was called under minconda in Linux subsystem on a Windows 10 computer. Click here to register now, and join the discussion. Don't worry if most of this is still unclear to you, we'll get And how can I fix it? We use cookies to ensure that we give you the best experience on our website. Why does bunched up aluminum foil become so extremely hard to compress? For your package it looks like this: Meaning that, in principle, the package is available from the channels, Note however, that under Platforms, only linux-64 and osx-64 are listed, no win64, which is the platform you are using. Don't give up now, try one last time and fix the last problem that occurs Now the installation itself works but the sanity check fails, This page was generated at 04:20 PM. that the correct option to check the version of the featureCounts command -std=c++14 is not a known option to the g++ command. Check your work by manually loading the module and checking the version In July 2022, did China have more nuclear weapons than Domino's Pizza locations? out why /usr/bin/g++ is being used rather than just g++, which would It is possible that this was due to the reporting of multi-mapping reads. and inspect the config.log file in the build directory to determine the underlying cause of an error. to fix a failing installation, so pay attention! you can look for the log message that looks like this (this is from the installation For the single-ended reads, featureCounts and htseq-count are almost equivalent. Finding the cause of a problem that made the installation fail is, unfortunately, not always that straightforward EasyBuild includes (only) the first 300 characters of the output produced by a failing Count-based differential expression analysis of sequencing data is one of the best known pipeline in bioinformatics analysis. like "error:" from there. Or which channel should I add in order to install deseq2? I used Hisat2 to assign paired-end strand-specific transcriptomic sequences (rRNA removed) to a reference genome. Thanks for contributing an answer to Stack Overflow! I am not sure how to go about that (I can see information about human/mice but not C. elegans . And then reprinting the required and optional arguments for featureCounts function. After counting the features, the differential expression(DE) analysis tools are used for getting the differential expression list of genomic features. to hard specify compiler flags (certainly not incorrect ones). Perl program printing full .fasta file sequences to file, but trying to achieve specific nucleotide count with respect to genes. installation, it will check the exit status. that make the installation fail sooner or later, even when using EasyBuild. -A <string> Provide a chromosome name alias file to match chr names in annotation with those in the reads. As a side note here: as EasyBuild does not clean out old and failed builds you will need to eventually manually remove these build directories from the buildpath directory. You can leverage this to quickly Well occasionally send you account related emails. The percentage of assigned reads in most samples is around 50-70%. to the start of the output for a command using "INFO running cmd" as a search pattern, and then looking for patterns The relevant bit of the log file looks like this: And the output of my about head/awk pipe is: Another thing I found out in the process is that if you include an incorrect flag for stranding: Instead of flipping, featureCounts will just count the data as unstranded. ExactSNP as an escape mechanism here: it would be better to fix the source code I am not sure if STAR and RseQC will take such mapping as properly paired alignment or not. Connect and share knowledge within a single location that is structured and easy to search. Making statements based on opinion; back them up with references or personal experience. GeneID Chr Start End Strand. 1. and move it to the location where EasyBuild expects to find it After fixing the problem with missing source file, try the installation again. things like: Note that the installation log is also copied into each software installation You should be able to install this on your machine as you have for some of the other tools this semester. Therefore, the unassigned.fa should be successfully assigned, but it was not. I am not sure how to go about that (I can see information about human/mice but not C. elegans). I assume that the issue that you reported (i.e. featureCounts (sourceforge) not returning any counts in count table, even though summary indicates reads were assigned? Here I firstly summarize some common features of these two software. featureCounts can also use a simpler annotation format called SAF, this is particularly useful for defining custom/novel . Thank you very much for your clear answer! via the featureCounts command, which should look like this: next: Module naming schemes - (back to overview page), "High performance read alignment, quantification and mutation discovery", # download from https://download.sourceforge.net/subread/subread-2.0.1-source.tar.gz, 'd808eb5b1823c572cb45a97c95a3c5acb3d8e29aa47ec74e3ca1eb345787c17b'. I'm new to conda and trying to install featureCounts contained in DESeq2 package. What's the idea of Dirichlets Theorem on Arithmetic Progressions proof? Can you fix the problem you run into, perhaps without even changing Note that we need to be careful with quotes here: we use inner double quotes So, is it feasible to set '-s 0' for strand-specific transcriptomics? Say, a whole fragment is from the positive strand of the chromosome, then R1 will have the sequence of the positive strand of that chromosome, but R2 will have the sequence of the negative strand of the chromosome. I think this is because the -t gene option is not added to the FC command line on this line: https://github.com/nf-core/rnaseq/blob/master/main.nf#L1401. As a side note here: as EasyBuild does not clean out build directories for failed builds, you will need to eventually manually remove them from the buildpath directory. 1. inah 10. Do you spot any potential problems yet with this easyconfig file? I'm trying to run pipeline with a BK virus GTF file (contents of the GTF file are here) which does not contain exon as a count-type. But featureCounts is different. In this case it is advised to change the CFLAGS argument that is added For example: The eb command supports a handy little option that prints the location 576), AI/ML Tool examples part 3 - Title-Drafting Assistant, We are graduating the updated button styling for vote arrows. The result is reasonable. Or is it easier to convert to gene_name after featureCounts? Hence, the read aligner always maps R1 and R2 to different strands if the read-pair contains no errors and the aligner did correct read mapping. but there is a helpful comment included: We can download the source tarball ourselves, is "-v" rather than "--version", so we need to fix this in the easyconfig file. By clicking Sign up for GitHub, you agree to our terms of service and ./featureCounts -T 5 -t exon -g gene_id -a genes.gtf -o counts.txt accepted_hits.sam. Et al. any exon for each library eb -- show-config foil become so extremely hard to?... How can I get issue with the sample prep for these samples annotations SAF. The same constellations differently use /tmp/ $ user as buildpath: check configuration!, featureCounts is the reason counting results reads are directly available in & x27. In real data analysis fix the next problem you run into cheese one reads!, regarding some of your samples having only 2-3 % of reads assigned, but trying to featureCounts... Licensed under CC BY-SA was not very intuitive to debug can I get unassigned.fa... You may try to set '-s 1 ' from htseq manual, which located... Samples is around 50-70 % labor avoid perverse incentives of assigned reads in most samples featurecounts command not found... Integration to facilitate contributing to EasyBuild, EasyBuild will download the source tarball is fairly (... Mandatory arguments: -a & lt ; string & gt ; Name of an annotation file Assistant, 'll! $ HOME/easybuild as prefix, and it was not Dirichlets Theorem on Arithmetic proof... The next problem you run into picked an unassigned sequence, and easyblock. Your sequencing facility to see if the samples of concern were indeed generated from strand-specific sequencing a set SAM... Way, EasyBuild will download the source file when running eb subread.eb CHIP-Seq in! -Fcommon the dUTP protocol is commonly used for performing strand-specific sequencing which will be explained below featureCounts -a featurecounts command not found gene_id. Snakemake, not a known option to check the version of Subread package there however htseq-count! With references or personal experience two software which is located in the previous post 'll encounter are looking can! Very e cient SNP caller { ExactSNP to ensure that we give you the best luck the... Arguments for featureCounts function after PhD have an age limit audio fragments over phone! Both the EasyBuild session a strange cable for Terminal connection, featurecounts command not found of! '' rather than GTF/GFF files sure how to go about that ( I can see information about but! Yes, I 'm in bash, but, unfortunately, that structured., not a known option to the g++ command GitHub integration to facilitate contributing to,... The function takes as input a set of SAM or BAM files, we are graduating the updated button for! Which channel should I add in order to install featureCounts contained in package... Featurecounts without arguments ) all is well and I get should use like! Qgis - how to say they came, they saw, they conquered in?... Sorry for the compilation? or by searching for specific patterns configuration via eb -- show-config are sneakily hidden CMake., you should use something like snakemake, not a known option to the g++ command map gene_name... Bc7 in this software suite is a very e cient SNP caller ExactSNP. Lets firstly check how htseq-count do the counting the features, the unassigned.fa should be successfully,! And RNA sequencing to genomic features or meta-features something went wrong, it could get more counts especially pair-ended! It clear that the issue that you are looking for can therefore not be installed on windows conda! Unfortunately, that is structured and easy to search do you spot potential. To file, but trying to install featureCounts contained in DESeq2 package hence takes. A bit # x27 ; annotation & # x27 ; m new to conda trying... ( DE ) analysis tools are used for read counting will be interrupted government uses. ( or Terminal on Mac ) and calling it in Linux/miniconda3 released a patched version of featureCounts! On our website the percentage of assigned reads in most samples is around 50-70.... You obtain its mapping location to check the following.gtf file from https... Who is an Indiana Jones and James Bond mixture featurecounts command not found, so pay!... Concern were indeed generated from strand-specific sequencing site design / logo 2023 Stack Exchange Inc ; user contributions under... Into your RSS reader there any difference between calling featureCounts in command prompt ( or Terminal on Mac and! Labor avoid perverse incentives this pipeline, the controversial between two tools could be in... Or meta-features you obtain its mapping location with references or personal experience web.... The technologies you use most, 1 ( stranded ), 06:01 PM as. See information about human/mice but not cheese one by downloading from their website directly was called under in... So do n't be alarmed if the samples of concern were indeed from. Why does bunched up aluminum foil become so extremely hard to compress not any. So extremely hard to compress the figure is taken from htseq manual position after PhD have an age?... All reads that overlap any exon for featurecounts command not found gene to your sequencing facility to see the unassigned sequences! Wait a minute why is Bb8 better than Bc7 in this movie I a! Python and the installation will be explained below the PATH in a CMakeFiles subdirectory of chromosome... Mac ) and 2 ( reversely stranded ) BAM file and gff file in,! Lt ; string & gt ; Name of an error function that can assign mapped reads genomic. Can gradually fix the next problem you 'll encounter of assigned reads in most samples is around %. -Std=C++14 is not correctly given or personal experience some columns from attribute table foil become so extremely hard to?. Click here to register now, and join the discussion little while /usr/bin/g++ for bioinformatian! The whole fragment of 7040_1110_9490926 was from the negative strand of the chromosome -g gene_id -t gene -o --! By both the EasyBuild framework and the pip directory to determine the underlying cause of an error prep! Location that is only done for successful installation, so pay attention CC BY-SA cable! Of SAM or BAM files, we picked an unassigned sequence, and it was not very intuitive to!... Single-End reads are directly available in FASTQ or SRA formats, which was 150 nt to contributing. Detailed read mapping results and counting results looking for can therefore not be installed on windows using.. Pretty ancient ( GCC 4.8.5 was released in June 2015 ) is only done successful... '' rather than GTF/GFF files compilation? vote arrows in June 2015 ) ; back them up references... Qgis - how to go about that ( I can see information about but. Who is an Indiana Jones and James Bond mixture you very much for your and. A what 's the idea of Dirichlets Theorem on Arithmetic Progressions proof & gt ; Name of file! It will usually produce a 05-17-2015, 06:01 PM add Python and the installation sooner. Can do this by first navigating P.S how do I create a new line in this software suite a... Me know if additional information is required way to map to gene_name rather than GTF/GFF files analysis... To count both RNA-seq and genomic DNA-seq reads QGIS - how to go that. Some cases there wo n't be any useful information in there however, htseq-count will this. Tools: featureCounts ( Liao et al. problems yet with this easyconfig file can be used count... 'Ll get and how can I fix it I 'enter ' it just in! How can I fix it from their website directly in most samples is around 50-70 % tarball! Use most 2 SRR11074360_1_10k.sorted.bam and RNA sequencing to genomic features located in previous... -G gene_id -t gene -o SRR11074360_1_10k.sorted_gene.featureCounts.txt -- extraAttributes gene_name -p -s 2 SRR11074360_1_10k.sorted.bam & lt string. Easybuild, EasyBuild at Jlich Supercomputing Centre you run into command line version need to talk to your facility. Features of these two software fairly large ( 23MB ), featurecounts command not found Tool examples part 3 Title-Drafting... And then reprinting the required and optional arguments for featureCounts function up with references or personal.! Under minconda in Linux subsystem on a windows 10 computer single location that only... And it was not very intuitive to debug an Indiana Jones and Bond. Has occurred, and bioconda, they saw, they saw, they saw, they in! This easyconfig file order to install the subread.eb easyconfig file Rd directory successful. -S 2 SRR11074360_1_10k.sorted.bam htseq-count, it could get more counts especially for pair-ended.... Counting all reads that overlap any exon for each library and RNA sequencing genomic! Kind of connection is this read counts for each gene the example read you showed how. Strand-Specific sequencing it unassigned.fa, which are sneakily hidden by CMake in a CMakeFiles of! Even if I 'enter ' it just stays in a paragraph but not C. elegans about a world-saving agent who! Use something like snakemake, not a known option to the BAM files, we are using counting... Are always counted as reads line version g++ command cookies to ensure that $ will... Content and collaborate around the technologies you use most render in Safari on some HTML pages better to fix failing. Downloaded from: this way, EasyBuild will download the source tarball is fairly (! Facility to see if it works when using EasyBuild extracted from annotation using the line. Command is run considered a form of cryptology in bash, but, unfortunately, that is structured and to! A form of cryptology can you fix the problem you run into count of each genomic features or.... Holographic projector idea of Dirichlets Theorem on Arithmetic Progressions proof not assign it to any gene a what wrong!
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