Closed reisingerf closed 6 years ago
Hi @reisingerf, this column must remain unique in the sample sheet as per the Illumina specification, however, this library does allow redundant Sample_ID
s only if the lanes are different (key Lane
).
At a minimum, the one column that is universally required is Sample_ID, which provides a unique string identifier for each sample.
Why, in your example, are you not using the Lane
key?
Would you be willing to give me more background on why you cannot give unique Sample_IDs
to these samples?
Also, for curiosity's sake, do you have any references from 10X I can look at?
Here are the current validations performed when a sample is added to the sample sheet:
I have seen the specs from Illumina, but it does not explicitly say that Sample_IDs can't be duplicated. It only says they have to uniquely identify a sample. E.g. in our case we want to demultiplex several lanes and indexes into the same FASTQ file (Sample_ID). We are using 10X genomics libraries, each sample is indexed with 4 indexes and run across 4 lanes. Our aim is to get a SINGLE FASTQ file per-read per-sample.
To pass validation you need to specify the column Lane
for each entry in your Sample Sheet.
Is that in conformance with the tools you are using from 10X? If not do send me a link.
So make it like this:
Sample_ID,Sample_Name,Lane,Sample_Plate,Sample_Well,Index_Plate_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description
PRJ180538_VPH20T,1,,,,,SI-GA-G6_1,CTGACGCG,,,,
PRJ180538_VPH20T,2,,,,,SI-GA-G6_2,GGTCGTAC,,,,
PRJ180538_VPH20T,3,,,,,SI-GA-G6_3,TCCTTCTT,,,,
PRJ180538_VPH20T,4,,,,,SI-GA-G6_4,AAAGAAGA,,,,
I don’t think that’ll work (but I am not the expert on this). As far as I understand the demultiplexing works across all lanes if no Lane column is given. If we where to use the Lane column, we’d have to use 16 lines (4 indexes over 4 lanes).
Am I missing something?
On 8 Aug 2018, at 15:09, Clint Valentine notifications@github.com<mailto:notifications@github.com> wrote:
So make it like this:
Sample_ID,Sample_Name,Lane,Sample_Plate,Sample_Well,Index_Plate_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description PRJ180538_VPH20T,1,,,,,SI-GA-G6_1,CTGACGCG,,,, PRJ180538_VPH20T,2,,,,,SI-GA-G6_2,GGTCGTAC,,,, PRJ180538_VPH20T,3,,,,,SI-GA-G6_3,TCCTTCTT,,,, PRJ180538_VPH20T,4,,,,,SI-GA-G6_4,AAAGAAGA,,,,
— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/clintval/sample-sheet/issues/53#issuecomment-411286971, or mute the threadhttps://github.com/notifications/unsubscribe-auth/AebZc-aeeI1Ih4oLnDyT_p20eQZjE7z9ks5uOnJ_gaJpZM4VzQhS.
I think I follow (yes you would have to specify 16 times under the Illumina spec.) Which demultiplexor or pipeline are you using? Can you provide me with docs?
We are using bcl2fastq with the —no-lane-splitting option.
On 8 Aug 2018, at 15:25, Clint Valentine notifications@github.com<mailto:notifications@github.com> wrote:
I think I follow (yes you would have to specify 16 times under the Illumina spec.) Which demultiplexor or pipeline are you using? Can you provide me with docs?
— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/clintval/sample-sheet/issues/53#issuecomment-411289083, or mute the threadhttps://github.com/notifications/unsubscribe-auth/AebZc3iU9OuaIlpE4djf6SFw0HTbGzmAks5uOnYkgaJpZM4VzQhS.
FYI, even the 10X demultiplexor requires specifying a Lane
if you want to demultiplex per lane:
https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/mkfastq
In order to help you further debug. I will need two things:
bcl2fastq
commandThanks! Hope I can help.
I will compile an example and get back to you. Thanks for your help!
Here is our SampleSheet.csv
[Header],,,,,,,,,,
IEMFileVersion,5,,,,,,,,,
Experiment Name,Tsqn180801,,,,,,,,,
Date,3/08/2018,,,,,,,,,
Workflow,GenerateFASTQ,,,,,,,,,
Application,NovaSeq FASTQ Only,,,,,,,,,
Instrument Type,NovaSeq,,,,,,,,,
Assay,TruSeq Nano DNA,,,,,,,,,
Index Adapters,IDT-ILMN TruSeq DNA UD Indexes (96 Indexes),,,,,,,,,
Description,Tsqn180801,,,,,,,,,
Chemistry,Amplicon,,,,,,,,,
,,,,,,,,,,
[Reads],,,,,,,,,,
151,,,,,,,,,,
151,,,,,,,,,,
,,,,,,,,,,
[Settings],,,,,,,,,,
Adapter,AGATCGGAAGAGCACACGTCTGAACTCCAGTCA,,,,,,,,,
AdapterRead2,AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT,,,,,,,,,
,,,,,,,,,,
[Data],,,,,,,,,,
Sample_ID,Sample_Name,Sample_Plate,Sample_Well,Index_Plate_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description
PRJ180538_VPH20T,,,,,SI-GA-G6_1,CTGACGCG,,,,
PRJ180538_VPH20T,,,,,SI-GA-G6_2,GGTCGTAC,,,,
PRJ180538_VPH20T,,,,,SI-GA-G6_3,TCCTTCTT,,,,
PRJ180538_VPH20T,,,,,SI-GA-G6_4,AAAGAAGA,,,,
PRJ180539_VCBPH5T,,,,,SI-GA-G7_1,GGTATGCA,,,,
PRJ180539_VCBPH5T,,,,,SI-GA-G7_2,CTCGAAAT,,,,
PRJ180539_VCBPH5T,,,,,SI-GA-G7_3,ACACCTTC,,,,
PRJ180539_VCBPH5T,,,,,SI-GA-G7_4,TAGTGCGG,,,,
PRJ180540_VCBP14T,,,,,SI-GA-G8_1,TATGAGCT,,,,
PRJ180540_VCBP14T,,,,,SI-GA-G8_2,CCGATAGC,,,,
PRJ180540_VCBP14T,,,,,SI-GA-G8_3,ATACCCAA,,,,
PRJ180540_VCBP14T,,,,,SI-GA-G8_4,GGCTGTTG,,,,
PRJ180541_VPH8T,,,,,SI-GA-G9_1,TAGGACGT,,,,
PRJ180541_VPH8T,,,,,SI-GA-G9_2,ATCCCACA,,,,
PRJ180541_VPH8T,,,,,SI-GA-G9_3,GGAATGTC,,,,
PRJ180541_VPH8T,,,,,SI-GA-G9_4,CCTTGTAG,,,,
PRJ180542_VPH23T,,,,,SI-GA-G10_1,TCGCCAGC,,,,
PRJ180542_VPH23T,,,,,SI-GA-G10_2,AATGTTAG,,,,
PRJ180542_VPH23T,,,,,SI-GA-G10_3,CGATAGCT,,,,
PRJ180542_VPH23T,,,,,SI-GA-G10_4,GTCAGCTA,,,,
PRJ180543_VPH36T,,,,,SI-GA-G11_1,TTATCGTT,,,,
PRJ180543_VPH36T,,,,,SI-GA-G11_2,AGCAGAGC,,,,
PRJ180543_VPH36T,,,,,SI-GA-G11_3,CATCTCCA,,,,
PRJ180543_VPH36T,,,,,SI-GA-G11_4,GCGGATAG,,,,
PRJ180544_PGL3,,,,,SI-GA-G12_1,ATTCTAAG,,,,
PRJ180544_PGL3,,,,,SI-GA-G12_2,CCCGATTA,,,,
PRJ180544_PGL3,,,,,SI-GA-G12_3,TGGAGGCT,,,,
PRJ180544_PGL3,,,,,SI-GA-G12_4,GAATCCGC,,,,
PRJ180545_LSI_noIAA,,,,,SI-GA-F3_1,TTCAGGTG,,,,
PRJ180545_LSI_noIAA,,,,,SI-GA-F3_2,ACGGACAT,,,,
PRJ180545_LSI_noIAA,,,,,SI-GA-F3_3,GATCTTGA,,,,
PRJ180545_LSI_noIAA,,,,,SI-GA-F3_4,CGATCACC,,,,
PRJ180546_LSI_IAA,,,,,SI-GA-F4_1,CCCAATAG,,,,
PRJ180546_LSI_IAA,,,,,SI-GA-F4_2,GTGTCGCT,,,,
PRJ180546_LSI_IAA,,,,,SI-GA-F4_3,AGAGTCGC,,,,
PRJ180546_LSI_IAA,,,,,SI-GA-F4_4,TATCGATA,,,,
PRJ180547_LL_30,,,,,SI-GA-F1_1,GTTGCAGC,,,,
PRJ180547_LL_30,,,,,SI-GA-F1_2,TGGAATTA,,,,
PRJ180547_LL_30,,,,,SI-GA-F1_3,CAATGGAG,,,,
PRJ180547_LL_30,,,,,SI-GA-F1_4,ACCCTCCT,,,,
PRJ180548_LL_38,,,,,SI-GA-F2_1,TTTACATG,,,,
PRJ180548_LL_38,,,,,SI-GA-F2_2,CGCGATAC,,,,
PRJ180548_LL_38,,,,,SI-GA-F2_3,ACGCGGGT,,,,
PRJ180548_LL_38,,,,,SI-GA-F2_4,GAATTCCA,,,,
And we use a command like this:
bcl2fastq -R /novaseq/runfolder --sample-sheet /novaseq/runfolder/SampleSheet.csv -o /bcl2fastq_output/runfolder --create-fastq-for-index-reads --minimum-trimmed-read-length=8 --mask-short-adapter-reads=8 --ignore-missing-positions --ignore-missing-controls --ignore-missing-filter --ignore-missing-bcls --no-lane-splitting
Thanks. Quick question. Does IEM allow you to make that sample sheet? Does bcl2fastq
accept it?
Just installed IEM and confirmed this is an allowable sample sheet!
I haven't checked with IEM, but we used that sample sheet with bcl2fastq successfully. (if it wouldn't work for us I would not request the change ;) )
Bug confirmed, thanks @reisingerf, I've misinterpreted the spec.
No worries! I initially was confused by the spec as well. I only realised due to our new requirement.
This library now allows adding equivalent samples, with a warning. See the docs for add_sample()
here for a way to turn-off that warning:
http://sample-sheet.readthedocs.io/sample_sheet.html#sample_sheet.SampleSheet.add_sample
I have it on my radar to do more research FYI.
Hi Clint,
we had an issue before (#32) where the same
Sample_ID
caused issues, even if the lanes were different.Now we are having the same issue, but without specifying lanes. This time it's 10X data, which you mentioned briefly in the previous issue.
Basically we would like to merge across lanes and indexes and the corresponding sample sheets end up having the same
Sample_ID
across multipleData
lines.An example:
Here we merge across the four indexes and all lanes. However, your library does not currently allow this.
Could you allow duplicated
Sample_ID
s or recommend an alternative approach?Thanks! Florian