When the cellranger mkfastq or cellranger count pipelines fail, they will automatically generate a "debug tarball" that contains the logs and metadata generated by the pipestance leading up to failure. This file, named sampleid. You may also use the cellranger upload command to send the tarball to 10x:. If you wish to troubleshoot a pipeline failure yourself, it is important to identify if you are experiencing a preflight failurean in-flight failureor an alert.
Preflight failures are the most common and are the result of invalid input data or runtime parameters. Because they occur before the pipeline actually runs, there will be no pipeline output and the error is reported directly to your terminal. Common preflight failures include failing to install bcl2fastq. In-flight failures are generally the result of factors external to the pipeline such as running out of system memory or disk space.
Different stages may fail in different ways so the specific error messages vary widely. There are a few important files that are saved to your pipeline output directory which, by default, is named according to the flowcell serial number for cellranger mkfastq e. A more detailed description of the pipeline output directory and its contents is given in the Pipestance Structure page. Once you have determined the reason for failure and are ready to continue running the pipeline, you can typically issue the same cellranger command to continue execution of the pipestance from the stage that originally failed.
When running cellranger antique pop up camper or cellranger countit will detect if its intended output directory already exists.
If it does, this existing pipeline output directory will be treated as an incomplete pipestance and resume execution. This feature allows pipelines to be stopped and resumed with great flexibility, but it can also result in errors such as:.
Alerts are generally the result of factors inherent in library preparation and sequencing instead of software. Alerts do not affect the operation of the pipeline, but they do highlight potential causes for abnormal or missing data. WARN alerts indicate that some parameter is suboptimal, but there may still be useful data in the pipeline output.
ERROR alerts indicate a major issue, and there is unlikely to usable results in the output. For example, running cellranger on a human sample with a human and mouse reference will likely result in an alert indicating low read alignment quality:. These WARN alerts are not indicative of a lost sequencing run, and re-running cellranger with the proper human reference would likely not return this alert.
For example, a low-quality library may result in the following alerts:. The presence of these ERROR alerts indicate that results that are output by this pipestance are likely dubious. What is Feature Barcoding? What is Loupe Browser? Download Link Installing Loupe Browser.
Bioinformatics Stack Exchange is a question and answer site for researchers, developers, students, teachers, and end users interested in bioinformatics. It only takes a minute to sign up. The issue was that I supplied wrong indexes during fastq files generation, however, they were generated successfully, and only the next step failed with not that informative error.
Sign up to join this community. The best answers are voted up and rise to the top. Home Questions Tags Users Unanswered. Asked 1 year, 11 months ago. Active 1 year, 11 months ago. Viewed times. This can happen if not enough reads originate from the given reference. Please verify your choice of reference or explicitly specify the chemistry via the --chemistry argument What is going wrong here?
Single-cell RNA sequencing (Cell Ranger)
How could I correct it? Nikita Vlasenko Nikita Vlasenko 2, 8 8 silver badges 25 25 bronze badges. Then, because we got bad results after running cellranger count 'bad' means biologically not what we want to see. It would mean that the biology was done in the wrong waywe transferred the raw data again, generated fastq files again and somehow now I am having this issue for the first replica, but not for the second one which ran successfully.
I am wondering where in this chain of actions I did a mistake. Can it be that the data got corrupted while I was transferring it, however, fastq generation worked still? Active Oldest Votes. Sign up or log in Sign up using Google. Sign up using Facebook.For a few days I have been trying to convert. Which on its own is not that hard. The real problem is reproducing the results. I am using a bamfile which already has a.
The bam file has nearly million reads with a MAPQ value of where the matrix. Also, GeneRanger does output the bamfile with their reads annotated, but not all of them. Of the million reads, only million actually have a gene in GN:Z: while the other million reads do not.
When I annotated the other million reads, the amount of total genes I find is pretty similar to what the matrix. The problem is that the counts per cell do not match at all. I get cells with 50 thousand reads if I convert the entire bam file to.
The amount of cells are pretty similar though. Now the first thing I thought was, they must be removing duplicates. However, if I look at all NH:i:1 entries, it exceeds way more than 30 million reads and the cells per gene do totally not scale when loading it into scanpy, some genes even have less cells eventhough there are more reads. Also looking at their xf:i: it does not make sense. This bam file has xf:i:0, xf:i, xf:i and xf:i And when statting the amount of reads per xf:i entry, I don't get any entry close to 30 million reads.
Furthermore, other bam files I loaded online do not even have xf:i:. I also do not think this is a phred score issue, as it should have been filtered out before alignment. What could this be? How can I identify the 30 million reads that CellRanger used to construct the matrix. I was thinking of trying FeatureCounts, but there still remains a problem if you want to get data such as SNPs from the bam file, if you don't know which reads to use and which not. There are whitelists of UMI indexes that 10x uses.
Have you looked into that angle? Their tech support is also pretty responsive so I suggest that you also contact them. Please come back and post their explanation when you hear back. Althought I did just find out that my problem is probably that I did not correct for UMIs, so gonna rerun my script and give an update later. I think CellRanger also removes any read where the Phred score of the UMI or cell barcode is below a threshold, althuogh I can't remember what that threshold is.The Cell Ranger software strives to maintain compatibility with common analysis tools by using standard output file formats whenever possible.
For example, the barcoded BAM files can be viewed in standard genome browsers such as IGV to verify alignment quality and other features. The Chromium-specific data, including cellular and molecular barcodes, can be accessed via any third-party tools or scripts that can parse the additional elements utilized by Cell Ranger.
All pipelines produce all of their output in a single pipeline output directorywhose name depends on the pipeline:. For example, a typical cellranger count may look like:. More information about the contents of the pipeline output directory can be found in the Pipestance Structure page. What is Feature Barcoding? What is Loupe Browser? Download Link Installing Loupe Browser.
After incubation, the GEMs are broken and the pooled fractions are recovered. Silane magnetic beads are used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. R1 read 1 primer sequence are added to the molecules during GEM incubation. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification.Advanced Sequencing Technologies 2015 - 10X Genomics - Rob Tarbox
Sample index sequences are incorporated as the i7 index read. Read 1 and Read 2 are standard Illumina sequencing primer sites used in paired-end sequencing. The final structure of library are shown below, in which 10X barcode specifies cell and randomer specifies transcript. The Cell Ranger workflow always starts with running cellranger mkfastq on each flowcell. The subsequent steps vary depending on how many samples, libraries and flowcells you have. We will describe them in order of increasing complexity:.
Single Sample, Library, and Flowcell is the most basic case. You have a single biological sample, which was prepared into a single library, and then sequenced on a single flowcell.
Follow the steps in Multi-Flowcell Samples to combine them in a single cellranger count run. One Sample, Multiple Libraries If you prepared multiple libraries from the same sample technical replicates, for examplethen each one should be run through a separate instance of cellranger count. Once those are completed, you can perform a combined analysis using cellranger aggras described in Multi-Library Aggregation.
Then you can combine them all in a single call to cellranger aggr. In this section, we uses the tiny-bcl example sequencing run as example. This will create a new subdirectory called cellranger-tiny-bcl Note that you can specify a 10x sample index set in the index column of the Data section:. In this example, only reads from lane 5 will be used. To search for the given sample index across all lanes, omit the lanes column entirely.
Before running the pipeline, we need to load cellrange by applying module load.Cell Ranger is delivered as a single, self-contained tar file that can be unpacked anywhere on your system. It bundles all of its required software dependencies, which are pre-compiled to run on a wide range of Linux distributions. For convenience, the reference data package required for Cell Ranger is provided as a separate download. Step 1 — Download and unpack the Cell Ranger file in any location.
This unpacks Cell Ranger, its dependencies, and the cellranger script into a new directory called cellranger Step 2 — Download and unpack any of the reference data files in a convenient location:. This creates a new directory called refdata-cellranger-GRCh Each reference contains a set of pre-generated indices and other data required by Cell Ranger.
This will allow you to invoke the cellranger command. Next, please run the bundled site check script and send the output to 10x. If requested, we will review the information to ensure that Cell Ranger will run smoothly once you have generated your own Chromium data.
Assuming you have installed Cell Ranger as described above, please run the following commands:. If you plan to run Cell Ranger on a clusterplease run and send us the output twice, once on a submit host and once on a cluster node. If your system does not have direct Internet connectivity, please send the output files as attachments to support 10xgenomics. To ensure that the cellranger pipeline is installed correctly, use cellranger testrun.
This test can take up to 60 minutes on a sixteen-core workstation. This tiny. It is generated whether the pipeline succeeds or fails. If the pipeline fails and you need troubleshooting assistance, you can send this file directly to us from the command line:.
If your system does not have direct Internet connectivity, you can also send the file as an attachment to support 10xgenomics.
What is Feature Barcoding?
What is Loupe Browser? Download Link Installing Loupe Browser. Site Check Script Next, please run the bundled site check script and send the output to 10x. Verify Installation To ensure that the cellranger pipeline is installed correctly, use cellranger testrun. All rights reserved. Pipestance completed successfully!I can't get cellranger count work.
It doesn't give errors but nothing happens and it takes me back to the command line. It only shows this:. I am running into the exact same problem.
Test run was successful etc. ATpoint did you ever figure out the issue? Verify Installation. Okay I figured it out for mine I think. So the "samplename" portion of the fastq can't have underscores. Let me know if that works for you. How is the software supposed to understand which sample you want to look at with "pbmc1k"?
Log In. Welcome to Biostar! Please log in to add an answer. I am trying to align 10X datasets using cellranger.
If someone has already manage to run cellRanger with Slurm, maybe you can help me : Until now, I Hello, Everyone, I cannot install cellranger. Thanks in advance for any help! According to their al Hi, I am new in single-cell RNA-seq. I got a library with a mixture of human and mouse cells. Hi All, I am working on a single cell rnaseq dataset for two conditions.
Hi i have a quick question, i have few aligned bam files from single cell RNA Seq data. I want to I don't have to do anything with DNase data Can anyone who have experience with analysis of Novogene fastq files via Cellranger Count Hi, I use cellranger mkref to build the customized mm10 genome reference. But it always showes e