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Old 04-10-2015, 02:36 AM   #1
paumarc
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Location: Zagreb

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Default Testing bcl2fast

Hello everybody!

I'm new at the NextSeq world, my lab just bought a new machine some weeks ago and now I will be responsible to set up all the environment to perform different bioinformatics analysis

The first i did was compile bcl2fastq in our cluster, I had no problem with it. Now i would like learn how does it works


I found some bcl files at the piccard GitHub https://github.com/broadinstitute/picard

but I'm not sure that I have all the archived required here, for example i do not have the SampleSheet file

so my questions are:

1- How do I can execute the bcl2fastq script. Do i have enough with

bcl2fastq --input-dir xxxx --ouput-dir xxxx

2 which files should i place to the input-dir? Do I have enougt with the bcl file or do i have to place some other files there? What if i don't have the SampleSheet file?

p
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Old 04-10-2015, 03:06 AM   #2
GenoMax
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Which sequencer are you getting? bcl2fastq v.2.16.x is required for NextSeq and Hiseq X data. If you are getting a MiSeq/HiSeq 1xxx/2xxx machine then you will want to use bcl2fastq v.1.8.4. The conversion programs included in these two versions are different and have different command syntax.

Quote from Illumina.

Quote:
The bcl2fastq2 v2.16 software can be used to convert BCL files from all Illumina sequencing systems running RTA version 2.0 and above. This is a change from the original information, which indicated that v2.16 could be used with all sequencers running RTA 1.18 and above: that information is no longer correct. For conversion of data generated on Illumina sequencing systems using versions of RTA before RTA 2.0, use bcl2fastq v1.8.4.
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Old 04-10-2015, 03:44 AM   #3
paumarc
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we have the Ilumina NextSeq 500 and our provider told me to use this software

http://support.illumina.com/content/...ware-v216.html

the point now is hos do i use it.

thanks
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Old 04-10-2015, 04:06 AM   #4
GenoMax
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You are using the right software.

You will need full data folder produced by the sequencer for a real test. I am not sure if you can download a full flowcell data folder to test the software. Check on BaseSpace.

Possible fastq conversion command will look like this. Demultiplexed data is going to be put in "Unaligned" directory. Following command is using 4 cores. There is no big benefit to going overboard with # of cores.

Code:
$ bcl2fastq -i /path_to/flowcell_ID/Data/Intensities/BaseCalls -o /path_to/flowcell_ID/Unaligned -r 4 -d 4 -p 4 -w 4
You can run

Code:
$ bcl2fastq -h
to get help on all possible command line options.

I believe you can specify a separate location for the samplesheet file with v.2.16. Previously SampleSheet.csv file had to be inside the /flowcell_ID/Data/Intensities/BaseCalls directory. That option would likely be specified with --samplesheet /path_to/samplesheet.csv addition to above command line (I have not tested the 2.16 version yet).

You can create an example samplesheet by using Illumina Experiment Manager (v.1.9) program that you can download from Illumina support site. This is a windows program.
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