Bcl2fastq conversion according to samples (demultiplexing)

Question

[1234anjalianjali1234]

hellow,

I am doing the bcl2fastq conversion of my RNA-Seq data (demultiplexing) but i am getting the results according to lane but not by samples.

my code:

sudo  bcl2fastq --input-dir ./Data/Intensities/BaseCalls -R ./ --no-lane-splitting --sample-sheet ./SampleSheet.csv

my samplesheet:

[Header],,,,
Date,2019-04-12,,,
Workflow,GenerateFASTQ,,,
Application,FASTQOnly,,,
Assay,TruSeq,,,
Description,,,,
Chemistry,,,,
,,,,
,,,,
[Reads],,,,
72,,,,
72,,,,
,,,,
,,,,
[Data],,,,
Lane,Sample_ID,Sample_Name,index,Sample_project
1,F0,0_DPI_MOCK,AGTCAA,Anjali
1,F2,2_DPI_MOCK,AGTTCC,Anjali
1,F5,5_DPI_MOCK,ATGTCA,Anjali
1,F7,7_DPI_MOCK,CCGTCC,Anjali
2,VF0,0_DPI_INFECTED,CGATGT,Anjali
2,VF2,2_DPI_INFECTED,TGACCA,Anjali
2,VF5,5_DPI_INFECTED,ACAGTG,Anjali
2,VF7,7_DPI_INFECTED,GCCAAT,Anjali
2,VF9,9_DPI_INFECTED,CAGATC,Anjali
2,VF10,10_DPI_INFECTED,CTTGTA,Anjali
3,F9,9_DPI_MOCK,GTCCGC,Anjali
3,F10,10_DPI_MOCK,GTGAAA,Anjali
3,F0,0_DPI_MOCK,AGTCAA,Anjali
3,F2,2_DPI_MOCK,AGTTCC,Anjali
4,CphiX,CONTROL,,Anjali
5,F5,5_DPI_MOCK,ATGTCA,Anjali
5,F7,7_DPI_MOCK,CCGTCC,Anjali
5,F9,9_DPI_MOCK,GTCCGC,Anjali
5,F10,10_DPI_MOCK,GTGAAA,Anjali
6,VF0,0_DPI_INFECTED,CGATGT,Anjali
6,VF2,2_DPI_INFECTED,TGACCA,Anjali
6,VF5,5_DPI_INFECTED,ACAGTG,Anjali
6,VF7,7_DPI_INFECTED,GCCAAT,Anjali
7,VF9,9_DPI_INFECTED,CAGATC,Anjali
7,VF10,10_DPI_INFECTED,CTTGTA,Anjali
7,VF0,0_DPI_INFECTED,CGATGT,Anjali
7,VF2,2_DPI_INFECTED,TGACCA,Anjali
8,VF5,5_DPI_INFECTED,ACAGTG,Anjali
8,VF7,7_DPI_INFECTED,GCCAAT,Anjali
8,VF9,9_DPI_INFECTED,CAGATC,Anjali
8,VF10,10_DPI_INFECTED,CTTGTA,Anjali

Thankyou

[Devon Ryan]

It doesn't help that your columns are in a non-standard order. It's quite likely that that broke things. They should be:

Lane,Sample_ID,Sample_Name,index,Sample_project

[1234anjalianjali1234]

Thankyou Devon,

I have updated my SampleSheet.csv

[sklages]

What did you get? What did you expect. There is a [data] section header missing. Did you try to find out what's going on from stdout output?

[1234anjalianjali1234]

Dear sklages, I am trying to convert the raw data (BCL files) from Illumina GAIIx into fastq files. I want the fastq files according to my samples, but i am getting the fastq file according to Lane (i.e. 16 fastq files of R1&R2 of each lane). Also i have updated my SampleSheet.csv and rerun the programme.

Thank you.

Answer 1

[GenoMax]

I am trying to convert the raw data (BCL files) from Illumina GAIIx into fastq files.

That does not sound right. GAIIx was one of the older Illumina sequencers and it never had BCL files. You must surely be using data from a new machine.

If you want to get the sample level files across all lanes, then use identical names in both Sample_ID and Sample_Name. e.g. 0_DPI_INFECTED if that sample is in pool run on all lanes.

6,0_DPI_INFECTED,0_DPI_INFECTED,CGATGT,Anjali

[1234anjalianjali1234]

Sorry if it doesn't sound right.

thank you for the advise I will try this.

But our samples were not pooled, we used biological triplicate for each sample. e.g. 0_DPI_INFECTED have 3 biological replicate in 3 different lane (lane- 2,6 and 7).

Sorry, I am not good in technical terms.

[Devon Ryan]

If your samples weren't pooled you couldn't have more than one on a lane.

[1234anjalianjali1234]

we have our sample in 3 different lanes for e.g. 0_DPI_INFECTED samples are in 3 different lane (lane- 2,6 and 7).

[GenoMax]

If those are biological replicates then you would want individual files for them from the three lanes.

Think of it this way. If Sample_1 ran in three lanes as a part of pool (technical replicates) then you can generate a single sample level file for that sample by setting identical Sample_1 name for those three lanes. If Sample_1_Rep1,Sample_1_Rep2,Sample_1_Rep3 ran in three separate lanes you would want to get separate files for those biological replicates.

[1234anjalianjali1234]

Yes, you are correct. This is what i want to do for each sample. Sorry, i was not very much clear in my question.

[GenoMax]

Then edit your sample sheet so it looks something like this (just showing one sample below)

2,0_DPI_INFECTED_Rep1,0_DPI_INFECTED_Rep1,CGATGT,Anjali
6,0_DPI_INFECTED_Rep2,0_DPI_INFECTED_Rep2,CGATGT,Anjali
7,0_DPI_INFECTED_Rep3,0_DPI_INFECTED_Rep3,CGATGT,Anjali

--no-lane-splitting is not useful if you don't have technical reps or if your sample did not run in more than one lane.

[1234anjalianjali1234]

I have updated my SampleSheet.csv file:

[Header],,,,
Date,2019-04-12,,,
Workflow,GenerateFASTQ,,,
Application,FASTQOnly,,,
Assay,TruSeq,,,
Description,,,,
Chemistry,,,,
,,,,
,,,,
[Reads],,,,
72,,,,
72,,,,
,,,,
,,,,
[Data],,,,
Lane,Sample_ID,Sample_Name,index,Sample_project
1,F0_rep_1,F0_rep_1,AGTCAA,Anjali
1,F2_rep_1,F2_rep_1,AGTTCC,Anjali
1,F5_rep_1,F5_rep_1,ATGTCA,Anjali
1,F7_rep_1,F7_rep_1,CCGTCC,Anjali
2,VF0_rep_1,VF0_rep_1,CGATGT,Anjali
2,VF2_rep_1,VF2_rep_1,TGACCA,Anjali
2,VF5_rep_1,VF5_rep_1,ACAGTG,Anjali
2,VF7_rep_1,VF7_rep_1,GCCAAT,Anjali
2,VF9_rep_1,VF9_rep_1,CAGATC,Anjali
2,VF10_rep_1,VF10_rep_1,CTTGTA,Anjali
3,F9_rep_1,F9_rep_1,GTCCGC,Anjali
3,F10_rep_1,F10_rep_1,GTGAAA,Anjali
3,F0_rep_2,F0_rep_2,AGTCAA,Anjali
3,F2_rep_2,F2_rep_2,AGTTCC,Anjali
4,CphiX,CphiX,,Anjali
5,F5_rep_2,F5_rep_2,ATGTCA,Anjali
5,F7_rep_2,F7_rep_2,CCGTCC,Anjali
5,F9_rep_2,F9_rep_2,GTCCGC,Anjali
5,F10_rep_2,F10_rep_2,GTGAAA,Anjali
6,VF0_rep_2,VF0_rep_2,CGATGT,Anjali
6,VF2_rep_2,VF2_rep_2,TGACCA,Anjali
6,VF5_rep_2,VF5_rep_2,ACAGTG,Anjali
6,VF7_rep_2,VF7_rep_2,GCCAAT,Anjali
7,VF9_rep_2,VF9_rep_2,CAGATC,Anjali
7,VF10_rep_2,VF10_rep_2,CTTGTA,Anjali
7,VF0_rep_3,VF0_rep_3,CGATGT,Anjali
7,VF2_rep_3,VF2_rep_3,TGACCA,Anjali
8,VF5_rep_3,VF5_rep_3,ACAGTG,Anjali
8,VF7_rep_3,VF7_rep_3,GCCAAT,Anjali
8,VF9_rep_3,VF9_rep_3,CAGATC,Anjali
8,VF10_rep_3,VF10_rep_3,CTTGTA,Anjali

But still not getting the desired result.

My code:

bcl2fastq --input-dir ./Data/Intensities/BaseCalls -R ./ --sample-sheet ./SampleSheet.csv --create-fastq-for-index-reads

[GenoMax]

But still not getting the desired result.

We can't read you mind nor see what files you are getting. Can you provide a listing of fastq files (ls -1 *.fastq.gz) and explain why that is not the result you want.

[1234anjalianjali1234]

output of

ls -1 *.fastq.gz

Undetermined_S0_L001_I1_001.fastq.gz
Undetermined_S0_L001_R1_001.fastq.gz
Undetermined_S0_L001_R2_001.fastq.gz
Undetermined_S0_L002_I1_001.fastq.gz
Undetermined_S0_L002_R1_001.fastq.gz
Undetermined_S0_L002_R2_001.fastq.gz
Undetermined_S0_L003_I1_001.fastq.gz
Undetermined_S0_L003_R1_001.fastq.gz
Undetermined_S0_L003_R2_001.fastq.gz
Undetermined_S0_L005_I1_001.fastq.gz
Undetermined_S0_L005_R1_001.fastq.gz
Undetermined_S0_L005_R2_001.fastq.gz
Undetermined_S0_L006_I1_001.fastq.gz
Undetermined_S0_L006_R1_001.fastq.gz
Undetermined_S0_L006_R2_001.fastq.gz
Undetermined_S0_L007_I1_001.fastq.gz
Undetermined_S0_L007_R1_001.fastq.gz
Undetermined_S0_L007_R2_001.fastq.gz
Undetermined_S0_L008_I1_001.fastq.gz
Undetermined_S0_L008_R1_001.fastq.gz
Undetermined_S0_L008_R2_001.fastq.gz

These are according to Lane but I want to get separate files for my samples.

[GenoMax]

Are you using Illumina expt manager to make the sample sheet? If not you may want to give that a try (Note: it is a windows only application).

Properly formatted SampleSheet.csv has more than the fields you have specified. They should have the following in Data part (this is a random example from Illumina Expt Manager) :

[Data]                          
Lane    Sample_ID   Sample_Name Sample_Plate    Sample_Well I7_Index_ID index   Sample_Project
1   Sample_1                AD002   CGATGT  
3   Sample_2                AD006   GCCAAT

[1234anjalianjali1234]

sorry, but i have confirmed that my data is from Illumina GAIIx sytem. IEM is not compatible with GAIIx. Thankyou

[Devon Ryan]

It's highly likely that whoever told you that was wrong. But in the unlikely event that they're correct you'll have to use an old version of bcl2fastq, since no recent versions are compatible with a GAIIx.

[Devon Ryan]

Are you getting the project directories as well? There should be an Anjali directory with a subdirectory for each library.

[1234anjalianjali1234]

Yes you are right, I will look into them. Thankyou