<p>So, we figured it out. If you delete everything until the <code>[Data]</code> tag in the sample sheet, leaving the <code>[Data]</code> tag, it starts working. Here is the full <code>sample_sheet.csv</code> that is working:</p>

<pre><code>[Data]
Lane,Sample_ID,Sample_Name,index,Sample_Project
1,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409
1,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409
1,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409
1,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409
1,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409
1,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409
1,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409
1,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409
2,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409
2,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409
2,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409
2,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409
2,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409
2,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409
2,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409
2,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409
3,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409
3,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409
3,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409
3,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409
3,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409
3,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409
3,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409
3,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409
4,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409
4,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409
4,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409
4,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409
4,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409
4,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409
4,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409
4,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409
</code></pre>

<p>It is very unfortunate and disappointing that <code>10X</code> website is generating wrong sample sheet. Still we got very low mapping rates and trying to figure out what went wrong.</p>

<blockquote>
  <p>Update</p>
</blockquote>

<p>I was supplying <code>--sample-sheet=</code> property, but it should be <code>--samplesheet=</code>. Now it is running fine (except for very low mapping rates) with all the headers on.</p>

<blockquote>
  <p>Update</p>
</blockquote>

<p>It turned out that we were given the wrong indexes, so we made a sample sheet using sample sheet generator again and even with <code>--samplesheet=</code> correct parameter it was not running. Convertion of <code>CLRF</code> with <code>perl</code> or <code>tr</code> did not help. However, concise format of the sample sheet worked out well which looks like the following:</p>

<pre><code>Lane,Sample,Index
1-4,17R,SI-GA-B8
1-4,19RL,SI-GA-B9
</code></pre>

<p>It needs to be supplied with <code>--csv=</code> parameter.</p>

CC BY-SA 3.0
2018-04-11T20:55:49.967Z
I found that if you copy and paste the 10X sheet directly from their website it inserts some odd characters on some of the blank lines.  I usually edit the file in vim and delete them.
GWW
CC BY-SA 3.0
2018-04-12T06:37:28.967Z
Related to @GWW, I suspect it was the ` ^M` lines (the ones with a space and then line end). Either way, I suggest that you email 10x support and report this issue. It should be easy for them to fix.
Devon Ryan
CC BY-SA 3.0
2018-04-12T17:08:12.637Z
It looks like it's a weird unicode character "U+00A0" / NO-BREAK SPACE.
GWW
CC BY-SA 3.0
2018-04-13T00:33:49.790Z
That's weird. I copied and pasted from their website and never had issues. But I also run `mac2unix`/`dos2unix` on every sample sheet, so maybe that is a sufficient fix.
burger
CC BY-SA 3.0
2018-04-11T20:38:44.463Z
2018-04-18T21:41:18.510Z
2018-04-18T21:41:18.510Z
Nikita Vlasenko
<p>Apart from supporting only Windows-style and Linux-style line endings and a restricted character set as mentioned above, the sample sheet <a href="https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/sequencing-sheet-format-specifications-technical-note-970-2017-004.pdf" rel="nofollow noreferrer">has to be in the correct encoding which is UTF-8</a> (or its subset US-ASCII).
Many command lines support these <a href="https://stackoverflow.com/questions/805418/how-to-find-encoding-of-a-file-via-script-on-linux">lookup</a> and <a href="https://stackoverflow.com/questions/64860/best-way-to-convert-text-files-between-character-sets">conversion</a> commands:</p>
<pre class="lang-bash prettyprint-override"><code># lookup
file -i my_sample_sheet.csv

# conversion
iconv -f ISO-8859-15 -t UTF-8 in.csv &gt; out.csv
</code></pre>

CC BY-SA 4.0
2020-08-26T21:45:26.303Z
2020-08-26T21:45:26.303Z
Tapper
<p>Also posted <a href="https://www.biostars.org/p/308123/" rel="nofollow noreferrer">on biostars</a>.</p>

<p>I am trying to use <code>cellranger</code> or <code>bcl2fastq</code> to convert the <code>.bcl</code> files that I got from single cell analysis run into <code>fastq</code> files for further analysis. I needed to generate <code>sample_sheet.csv</code> and so I used the following tool:</p>

<p><a href="https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/bcl2fastq-direct" rel="nofollow noreferrer">https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/bcl2fastq-direct</a></p>

<p>which I copy-pasted into a text file using <code>vim</code>. <code>sample_sheet.csv</code> looks like that:</p>

<pre><code>[Header]
EMFileVersion,4


[Reads]
26
8
98

[Data]
Lane,Sample_ID,Sample_Name,index,Sample_Project
1,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180406
1,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180406
1,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180406
1,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180406
1,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180406 
1,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180406
1,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180406
1,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180406
2,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180406
2,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180406
2,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180406
2,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180406
2,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180406
2,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180406
2,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180406
2,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180406
3,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180406
3,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180406
3,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180406
3,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180406
3,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180406
3,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180406
3,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180406
3,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180406
4,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180406
4,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180406
4,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180406
4,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180406
4,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180406
4,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180406
4,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180406
</code></pre>

<p>Then I ran <code>cellranger</code> using <code>bash</code> script:</p>

<pre><code>#!/bin/bash

 FLOWCELL_DIR="/scratch/nv4e/kipnis/180403_NB501830_0158_AHN3LLBGX5"
 OUTPUT_DIR="/scratch/nv4e/kipnis/fastq"
 SAMPLE_SHEET_PATH="/scratch/nv4e/kipnis/sample_sheet_2.csv"

cellranger mkfastq --id="AHN3LLBGX5" --run=${FLOWCELL_DIR} --csv=${SAMPLE_SHEET_PATH} --output-dir=${OUTPUT_DIR}
</code></pre>

<p>It fails with the following output:</p>

<p><a href="https://i.sstatic.net/coXGs.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/coXGs.png" alt="enter image description here"></a></p>

<p><code>_stderr</code> file shows:</p>

<p><a href="https://i.sstatic.net/ECDVI.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/ECDVI.png" alt="enter image description here"></a></p>

<p>and that my <code>sample_sheet.csv</code> can not be parsed:</p>

<p><a href="https://i.sstatic.net/NVNW6.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/NVNW6.png" alt="enter image description here"></a></p>

<p>It could be because <code>CLRF</code> needs to be converted into <code>LF</code>, but I tried two commands to do this unsuccessfully:</p>

<p><code>tr -d '\r' &lt; input &gt; output</code> and <code>perl -pi -e 's/\r\n/\n/g' input</code></p>

<p>From the following thread:</p>

<p><a href="https://unix.stackexchange.com/questions/277217/how-to-install-dos2unix-on-linux-without-root-access">https://unix.stackexchange.com/questions/277217/how-to-install-dos2unix-on-linux-without-root-access</a></p>

<p>The above commands successfully strip the <code>\r</code> from the line endings, but the error from bcl2fastq remains.</p>

<p>What am I doing wrong? Any suggestions would be greatly appreciated.</p>

<blockquote>
  <p>Update</p>
</blockquote>

<p>I looked into the folder that is generated when the <code>cellranger</code> runs and found two <code>.csv</code> files generated by the <code>cellranger</code>. One of them, <code>samplesheet.csv</code>, contains these <code>^M</code> characters that are shown in the error:</p>

<p><a href="https://i.sstatic.net/1FYBP.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/1FYBP.png" alt="enter image description here"></a></p>

<blockquote>
  <p>Update</p>
</blockquote>

<p>Maybe one of the issues was that I was submitting regular <code>.sh</code> file on the <code>SLURM</code> cluster. However, I wrote the right <code>.slurm</code> file and submitted it yesterday with <code>sbatch</code> command:</p>

<p><a href="https://i.sstatic.net/zjDEy.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/zjDEy.png" alt="enter image description here"></a></p>

<p>However, today I got the message in my email:</p>

<p><a href="https://i.sstatic.net/npZzE.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/npZzE.png" alt="enter image description here"></a></p>

<p>And neither <code>_stderr</code>, nor <code>generate_fastq_id.err</code> or <code>generate_fastq_id.out</code> files contain anything. I thought initially that I set not enough time and it timed out, however, it is not true: I set the time to <code>24h</code> and it has been running for just <code>13h</code> before failing. </p>

<blockquote>
  <p>Update</p>
</blockquote>

<p>I searched over all of the files in the generated folder with the <code>find</code> linux command and actually found the error files that show the same error as before: issue with the <code>sample_sheet.csv</code> file. This error file is <code>AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/BCL2FASTQ_WITH_SAMPLESHEET/fork0/chnk0-u179acba3d8/_stderr</code>.</p>

<p>So, right now the issue seems to be the following: I am feeding in the right <code>sample_sheet.csv</code> without the <code>^M</code> symbols, but <code>cellranger</code> transforms it into one that contains <code>^M</code> and then uses it giving the error. Above I see also <code>which: no configureBclToFastq.pl</code> and <code>ERROR: bcl2fastq::common::Exception: 2018-Apr-09 13:33:14: Inappropriate ioctl for device</code>, so maybe my input parameters are just wrong to <code>cellranger</code> and it has nothing to do with the <code>sample_sheet.csv</code>? </p>

<blockquote>
  <p>Update</p>
</blockquote>

<p>I tried to use <code>--sample-sheet</code> instead of <code>--csv</code> since I am feeding in complex sample sheet. No luck.</p>

<blockquote>
  <p>Update</p>
</blockquote>

<p>Here you can find all the error document in text:</p>

<blockquote>
  <p>which: no configureBclToFastq.pl in (/sfs/lustre/scratch/nv4e/cellranger/samtools_new/1.6:/sfs/lustre/scratch/nv4e/cellranger/cellranger-tiny-fastq/1.2.0:/sfs/lustre/scratch/nv4e/cellranger/cellranger-tiny-ref/1.2.0:/sfs/lustre/scratch/nv4e/cellranger/miniconda-cr-cs/4.3.21-miniconda-cr-cs-c9/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/lib/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/tenkit/lib/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/tenkit/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/bin:/sfs/lustre/scratch/nv4e/cellranger/lz4/v1.8.0:/sfs/lustre/scratch/nv4e/cellranger/martian-cs/2.3.2/bin:/sfs/lustre/scratch/nv4e/cellranger/STAR/5dda596:/sfs/lustre/scratch/nv4e/cellranger/samtools_new/1.6:/sfs/lustre/scratch/nv4e/cellranger/cellranger-tiny-fastq/1.2.0:/sfs/lustre/scratch/nv4e/cellranger/cellranger-tiny-ref/1.2.0:/sfs/lustre/scratch/nv4e/cellranger/miniconda-cr-cs/4.3.21-miniconda-cr-cs-c9/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/lib/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/tenkit/lib/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/tenkit/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/bin:/sfs/lustre/scratch/nv4e/cellranger/lz4/v1.8.0:/sfs/lustre/scratch/nv4e/cellranger/martian-cs/2.3.2/bin:/sfs/lustre/scratch/nv4e/cellranger/STAR/5dda596:/scratch/nv4e/cellranger:/scratch/nv4e/bcl2fastq/build/bin:/scratch/nv4e/spark/bin:/scratch/nv4e/scala/bin:/sfs/nfs/blue/nv4e/private/bin:/sfs/nfs/blue/nv4e/anaconda2/bin:/sfs/nfs/blue/nv4e/.local/bin:/usr/lib64/qt-3.3/bin:/usr/local/bin:/usr/bin:/usr/local/sbin:/usr/sbin:/opt/slurm/current/bin:/opt/slurm/current/sbin:/opt/singularity/current/bin:/opt/rci/bin:/opt/rci/sbin:/opt/nhc/current/sbin:/share/rci_apps/common/bin:/share/resources/HPCtools/)
  BCL to FASTQ file converter
  bcl2fastq v2.20.0.422
  Copyright (c) 2007-2017 Illumina, Inc.
  2018-04-09 18:31:29 [7fcdb39f37c0] Command-line invocation: bcl2fastq --minimum-trimmed-read-length 8 --mask-short-adapter-reads 8 --create-fastq-for-index-reads --ignore-missing-positions --ignore-missing-filter --ignore-missing-bcls --use-bases-mask=Y26,I8,Y98 -R /scratch/nv4e/kipnis/180403_NB501830_0158_AHN3LLBGX5 --output-dir=/scratch/nv4e/kipnis/AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/BCL2FASTQ_WITH_SAMPLESHEET/fork0/chnk0-u21f1cbe9bf/files/fastq_path --interop-dir=/scratch/nv4e/kipnis/AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/BCL2FASTQ_WITH_SAMPLESHEET/fork0/chnk0-u21f1cbe9bf/files/interop_path --sample-sheet=/scratch/nv4e/kipnis/AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/PREPARE_SAMPLESHEET/fork0/chnk0-u09e7cbe891/files/samplesheet.csv -p 6 -r 6 -w 6
  2018-04-09 18:31:29 [7fcdb39f37c0] INFO: Minimum log level: INFO
  2018-04-09 18:31:29 [7fcdb39f37c0] INFO: Sample sheet: '/scratch/nv4e/kipnis/AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/PREPARE_SAMPLESHEET/fork0/chnk0-u09e7cbe891/files/samplesheet.csv'
  2018-04-09 18:31:29 [7fcdb39f37c0] ERROR: bcl2fastq::common::Exception: 2018-Apr-09 18:31:29: Inappropriate ioctl for device (25): /scratch/nv4e/bcl2fastq/src/cxx/include/common/CsvGrammar.hpp(92): Throw in function bcl2fastq::common::CsvGrammarAttribute bcl2fastq::common::parseCsvData(Iterator, Iterator) [with Iterator = __gnu_cxx::__normal_iterator >; bcl2fastq::common::CsvGrammarAttribute = std::vector > >]
  Dynamic exception type: boost::exception_detail::clone_impl
  std::exception::what: Could not parse the CSV stream text:
   ^M
  ^M
  [Reads]^M
  26^M
  98^M
   ^M
  ^M
  [Data]^M
  Lane,Sample_ID,Sample_Name,index,Sample_Project,Original_Sample_ID^M
  1,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409,SI-GA-B4_1^M
  1,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409,SI-GA-B4_2^M
  1,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409,SI-GA-B4_3^M
  1,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409,SI-GA-B4_4^M
  1,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409,SI-GA-B5_1^M
  1,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409,SI-GA-B5_2^M
  1,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409,SI-GA-B5_3^M
  1,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409,SI-GA-B5_4^M
  2,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409,SI-GA-B4_1^M
  2,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409,SI-GA-B4_2^M
  2,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409,SI-GA-B4_3^M
  2,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409,SI-GA-B4_4^M
  2,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409,SI-GA-B5_1^M
  2,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409,SI-GA-B5_2^M
  2,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409,SI-GA-B5_3^M
  2,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409,SI-GA-B5_4^M
  3,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409,SI-GA-B4_1^M
  3,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409,SI-GA-B4_2^M
  3,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409,SI-GA-B4_3^M
  3,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409,SI-GA-B4_4^M
  3,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409,SI-GA-B5_1^M
  3,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409,SI-GA-B5_2^M
  3,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409,SI-GA-B5_3^M
  3,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409,SI-GA-B5_4^M
  4,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409,SI-GA-B4_1^M
  4,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409,SI-GA-B4_2^M
  4,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409,SI-GA-B4_3^M
  4,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409,SI-GA-B4_4^M
  4,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409,SI-GA-B5_1^M
  4,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409,SI-GA-B5_2^M
  4,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409,SI-GA-B5_3^M
  4,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409,SI-GA-B5_4^M</p>
</blockquote>

CC BY-SA 3.0
2018-04-08T22:28:45.723Z
What happens if you remove the duplicate emtpy lines (just keep one before each section)?
Devon Ryan
CC BY-SA 3.0
2018-04-08T23:45:25.330Z
There are no duplicate lines in the input file, but inside the generated `_stderr` file they are somehow present
Nikita Vlasenko
CC BY-SA 3.0
2018-04-09T09:00:26.293Z
Is that the output from `_stderr` after removing `\r`? Because the `^M` characters indicate a line break problem. If they go away after running the `tr`/`perl` command, then there's probably an additional problem
heathobrien
CC BY-SA 3.0
2018-04-09T17:49:14.977Z
Yes, it is after fixing the sample sheet.
Nikita Vlasenko
CC BY-SA 3.0
2018-04-10T09:58:11.990Z
Did you manage to install the configureBclToFastq.pl in the path as reported in the _stderr? BTW, if you make your error message as text it would be easier for us to search and quote specific parts of the error: CTRL+SHIFT+C and CTRL+SHIFT+V in terminal
llrs
CC BY-SA 3.0
2018-04-10T17:44:16.917Z
No, I have not yet tried installing it because I do not know how. Could you give any links of how to do that and where to get the file?
Nikita Vlasenko
CC BY-SA 3.0
2018-04-10T19:17:35.440Z
There's no reason to install the old bcl2fastq (the configureBclToFastq.pl that's mentioned). Can you directly run `bcl2fastq` with your sample sheet (i.e., without using cellranger)?
Devon Ryan
<scrnaseq><10x-genomics>
10X Illumina demultiplexing sample sheet issue
# 10X Illumina demultiplexing sample sheet issue

(Asked by: username_0 on 2018-04-08)

Also posted on biostars.
I am trying to use cellranger or bcl2fastq to convert the .bcl files that I got from single cell analysis run into fastq files for further analysis. I needed to generate sample_sheet.csv and so I used the following tool:
https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/bcl2fastq-direct
which I copy-pasted into a text file using vim. sample_sheet.csv looks like that:
[Header]
EMFileVersion,4


[Reads]
26
8
98

[Data]
Lane,Sample_ID,Sample_Name,index,Sample_Project
1,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180406
1,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180406
1,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180406
1,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180406
1,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180406 
1,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180406
1,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180406
1,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180406
2,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180406
2,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180406
2,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180406
2,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180406
2,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180406
2,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180406
2,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180406
2,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180406
3,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180406
3,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180406
3,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180406
3,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180406
3,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180406
3,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180406
3,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180406
3,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180406
4,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180406
4,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180406
4,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180406
4,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180406
4,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180406
4,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180406
4,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180406

Then I ran cellranger using bash script:
#!/bin/bash

 FLOWCELL_DIR="/scratch/nv4e/kipnis/180403_NB501830_0158_AHN3LLBGX5"
 OUTPUT_DIR="/scratch/nv4e/kipnis/fastq"
 SAMPLE_SHEET_PATH="/scratch/nv4e/kipnis/sample_sheet_2.csv"

cellranger mkfastq --id="AHN3LLBGX5" --run=${FLOWCELL_DIR} --csv=${SAMPLE_SHEET_PATH} --output-dir=${OUTPUT_DIR}

It fails with the following output:

_stderr file shows:

and that my sample_sheet.csv can not be parsed:

It could be because CLRF needs to be converted into LF, but I tried two commands to do this unsuccessfully:
tr -d '\r' < input > output and perl -pi -e 's/\r\n/\n/g' input
From the following thread:
https://unix.stackexchange.com/questions/277217/how-to-install-dos2unix-on-linux-without-root-access
The above commands successfully strip the \r from the line endings, but the error from bcl2fastq remains.
What am I doing wrong? Any suggestions would be greatly appreciated.

Update

I looked into the folder that is generated when the cellranger runs and found two .csv files generated by the cellranger. One of them, samplesheet.csv, contains these ^M characters that are shown in the error:


Update

Maybe one of the issues was that I was submitting regular .sh file on the SLURM cluster. However, I wrote the right .slurm file and submitted it yesterday with sbatch command:

However, today I got the message in my email:

And neither _stderr, nor generate_fastq_id.err or generate_fastq_id.out files contain anything. I thought initially that I set not enough time and it timed out, however, it is not true: I set the time to 24h and it has been running for just 13h before failing. 

Update

I searched over all of the files in the generated folder with the find linux command and actually found the error files that show the same error as before: issue with the sample_sheet.csv file. This error file is AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/BCL2FASTQ_WITH_SAMPLESHEET/fork0/chnk0-u179acba3d8/_stderr.
So, right now the issue seems to be the following: I am feeding in the right sample_sheet.csv without the ^M symbols, but cellranger transforms it into one that contains ^M and then uses it giving the error. Above I see also which: no configureBclToFastq.pl and ERROR: bcl2fastq::common::Exception: 2018-Apr-09 13:33:14: Inappropriate ioctl for device, so maybe my input parameters are just wrong to cellranger and it has nothing to do with the sample_sheet.csv? 

Update

I tried to use --sample-sheet instead of --csv since I am feeding in complex sample sheet. No luck.

Update

Here you can find all the error document in text:

which: no configureBclToFastq.pl in (/sfs/lustre/scratch/nv4e/cellranger/samtools_new/1.6:/sfs/lustre/scratch/nv4e/cellranger/cellranger-tiny-fastq/1.2.0:/sfs/lustre/scratch/nv4e/cellranger/cellranger-tiny-ref/1.2.0:/sfs/lustre/scratch/nv4e/cellranger/miniconda-cr-cs/4.3.21-miniconda-cr-cs-c9/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/lib/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/tenkit/lib/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/tenkit/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/bin:/sfs/lustre/scratch/nv4e/cellranger/lz4/v1.8.0:/sfs/lustre/scratch/nv4e/cellranger/martian-cs/2.3.2/bin:/sfs/lustre/scratch/nv4e/cellranger/STAR/5dda596:/sfs/lustre/scratch/nv4e/cellranger/samtools_new/1.6:/sfs/lustre/scratch/nv4e/cellranger/cellranger-tiny-fastq/1.2.0:/sfs/lustre/scratch/nv4e/cellranger/cellranger-tiny-ref/1.2.0:/sfs/lustre/scratch/nv4e/cellranger/miniconda-cr-cs/4.3.21-miniconda-cr-cs-c9/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/lib/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/tenkit/lib/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/tenkit/bin:/sfs/lustre/scratch/nv4e/cellranger/cellranger-cs/2.1.1/bin:/sfs/lustre/scratch/nv4e/cellranger/lz4/v1.8.0:/sfs/lustre/scratch/nv4e/cellranger/martian-cs/2.3.2/bin:/sfs/lustre/scratch/nv4e/cellranger/STAR/5dda596:/scratch/nv4e/cellranger:/scratch/nv4e/bcl2fastq/build/bin:/scratch/nv4e/spark/bin:/scratch/nv4e/scala/bin:/sfs/nfs/blue/nv4e/private/bin:/sfs/nfs/blue/nv4e/anaconda2/bin:/sfs/nfs/blue/nv4e/.local/bin:/usr/lib64/qt-3.3/bin:/usr/local/bin:/usr/bin:/usr/local/sbin:/usr/sbin:/opt/slurm/current/bin:/opt/slurm/current/sbin:/opt/singularity/current/bin:/opt/rci/bin:/opt/rci/sbin:/opt/nhc/current/sbin:/share/rci_apps/common/bin:/share/resources/HPCtools/)
  BCL to FASTQ file converter
  bcl2fastq v2.20.0.422
  Copyright (c) 2007-2017 Illumina, Inc.
  2018-04-09 18:31:29 [7fcdb39f37c0] Command-line invocation: bcl2fastq --minimum-trimmed-read-length 8 --mask-short-adapter-reads 8 --create-fastq-for-index-reads --ignore-missing-positions --ignore-missing-filter --ignore-missing-bcls --use-bases-mask=Y26,I8,Y98 -R /scratch/nv4e/kipnis/180403_NB501830_0158_AHN3LLBGX5 --output-dir=/scratch/nv4e/kipnis/AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/BCL2FASTQ_WITH_SAMPLESHEET/fork0/chnk0-u21f1cbe9bf/files/fastq_path --interop-dir=/scratch/nv4e/kipnis/AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/BCL2FASTQ_WITH_SAMPLESHEET/fork0/chnk0-u21f1cbe9bf/files/interop_path --sample-sheet=/scratch/nv4e/kipnis/AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/PREPARE_SAMPLESHEET/fork0/chnk0-u09e7cbe891/files/samplesheet.csv -p 6 -r 6 -w 6
  2018-04-09 18:31:29 [7fcdb39f37c0] INFO: Minimum log level: INFO
  2018-04-09 18:31:29 [7fcdb39f37c0] INFO: Sample sheet: '/scratch/nv4e/kipnis/AHN3LLBGX5/MAKE_FASTQS_CS/MAKE_FASTQS/PREPARE_SAMPLESHEET/fork0/chnk0-u09e7cbe891/files/samplesheet.csv'
  2018-04-09 18:31:29 [7fcdb39f37c0] ERROR: bcl2fastq::common::Exception: 2018-Apr-09 18:31:29: Inappropriate ioctl for device (25): /scratch/nv4e/bcl2fastq/src/cxx/include/common/CsvGrammar.hpp(92): Throw in function bcl2fastq::common::CsvGrammarAttribute bcl2fastq::common::parseCsvData(Iterator, Iterator) [with Iterator = __gnu_cxx::__normal_iterator >; bcl2fastq::common::CsvGrammarAttribute = std::vector > >]
  Dynamic exception type: boost::exception_detail::clone_impl
  std::exception::what: Could not parse the CSV stream text:
   ^M
  ^M
  [Reads]^M
  26^M
  98^M
   ^M
  ^M
  [Data]^M
  Lane,Sample_ID,Sample_Name,index,Sample_Project,Original_Sample_ID^M
  1,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409,SI-GA-B4_1^M
  1,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409,SI-GA-B4_2^M
  1,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409,SI-GA-B4_3^M
  1,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409,SI-GA-B4_4^M
  1,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409,SI-GA-B5_1^M
  1,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409,SI-GA-B5_2^M
  1,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409,SI-GA-B5_3^M
  1,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409,SI-GA-B5_4^M
  2,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409,SI-GA-B4_1^M
  2,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409,SI-GA-B4_2^M
  2,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409,SI-GA-B4_3^M
  2,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409,SI-GA-B4_4^M
  2,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409,SI-GA-B5_1^M
  2,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409,SI-GA-B5_2^M
  2,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409,SI-GA-B5_3^M
  2,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409,SI-GA-B5_4^M
  3,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409,SI-GA-B4_1^M
  3,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409,SI-GA-B4_2^M
  3,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409,SI-GA-B4_3^M
  3,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409,SI-GA-B4_4^M
  3,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409,SI-GA-B5_1^M
  3,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409,SI-GA-B5_2^M
  3,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409,SI-GA-B5_3^M
  3,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409,SI-GA-B5_4^M
  4,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409,SI-GA-B4_1^M
  4,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409,SI-GA-B4_2^M
  4,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409,SI-GA-B4_3^M
  4,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409,SI-GA-B4_4^M
  4,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409,SI-GA-B5_1^M
  4,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409,SI-GA-B5_2^M
  4,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409,SI-GA-B5_3^M
  4,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409,SI-GA-B5_4^M

**Tags:** scrnaseq, 10x-genomics
> **Comments:**
> **username_1**: What happens if you remove the duplicate emtpy lines (just keep one before each section)?  
> **username_0**: There are no duplicate lines in the input file, but inside the generated `_stderr` file they are somehow present  
> **username_2**: Is that the output from `_stderr` after removing `\r`? Because the `^M` characters indicate a line break problem. If they go away after running the `tr`/`perl` command, then there's probably an additional problem  
> **username_0**: Yes, it is after fixing the sample sheet.  
> **username_3**: Did you manage to install the configureBclToFastq.pl in the path as reported in the _stderr? BTW, if you make your error message as text it would be easier for us to search and quote specific parts of the error: CTRL+SHIFT+C and CTRL+SHIFT+V in terminal  
> **username_0**: No, I have not yet tried installing it because I do not know how. Could you give any links of how to do that and where to get the file?  
> **username_1**: There's no reason to install the old bcl2fastq (the configureBclToFastq.pl that's mentioned). Can you directly run `bcl2fastq` with your sample sheet (i.e., without using cellranger)?  

---

## 2 answers

### Answer by username_0 on 2018-04-11. Score: 3 | Accepted answer

So, we figured it out. If you delete everything until the [Data] tag in the sample sheet, leaving the [Data] tag, it starts working. Here is the full sample_sheet.csv that is working:
[Data]
Lane,Sample_ID,Sample_Name,index,Sample_Project
1,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409
1,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409
1,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409
1,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409
1,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409
1,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409
1,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409
1,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409
2,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409
2,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409
2,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409
2,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409
2,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409
2,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409
2,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409
2,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409
3,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409
3,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409
3,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409
3,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409
3,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409
3,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409
3,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409
3,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409
4,SI-GA-B4_1,17R,ACTTCATA,Chromium_20180409
4,SI-GA-B4_2,17R,GAGATGAC,Chromium_20180409
4,SI-GA-B4_3,17R,TGCCGTGG,Chromium_20180409
4,SI-GA-B4_4,17R,CTAGACCT,Chromium_20180409
4,SI-GA-B5_1,19RL,AATAATGG,Chromium_20180409
4,SI-GA-B5_2,19RL,CCAGGGCA,Chromium_20180409
4,SI-GA-B5_3,19RL,TGCCTCAT,Chromium_20180409
4,SI-GA-B5_4,19RL,GTGTCATC,Chromium_20180409

It is very unfortunate and disappointing that 10X website is generating wrong sample sheet. Still we got very low mapping rates and trying to figure out what went wrong.

Update

I was supplying --sample-sheet= property, but it should be --samplesheet=. Now it is running fine (except for very low mapping rates) with all the headers on.

Update

It turned out that we were given the wrong indexes, so we made a sample sheet using sample sheet generator again and even with --samplesheet= correct parameter it was not running. Convertion of CLRF with perl or tr did not help. However, concise format of the sample sheet worked out well which looks like the following:
Lane,Sample,Index
1-4,17R,SI-GA-B8
1-4,19RL,SI-GA-B9

It needs to be supplied with --csv= parameter.

> **Comments:**
> **username_4**: I found that if you copy and paste the 10X sheet directly from their website it inserts some odd characters on some of the blank lines.  I usually edit the file in vim and delete them.  
> **username_1**: Related to @GWW, I suspect it was the ` ^M` lines (the ones with a space and then line end). Either way, I suggest that you email 10x support and report this issue. It should be easy for them to fix.  
> **username_4**: It looks like it's a weird unicode character "U+00A0" / NO-BREAK SPACE.  
> **username_5**: That's weird. I copied and pasted from their website and never had issues. But I also run `mac2unix`/`dos2unix` on every sample sheet, so maybe that is a sufficient fix.  

---

### Answer by username_6 on 2020-08-26. Score: 0

Apart from supporting only Windows-style and Linux-style line endings and a restricted character set as mentioned above, the sample sheet has to be in the correct encoding which is UTF-8 (or its subset US-ASCII).
Many command lines support these lookup and conversion commands:
# lookup
file -i my_sample_sheet.csv

# conversion
iconv -f ISO-8859-15 -t UTF-8 in.csv > out.csv


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