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Old 03-06-2019, 11:27 AM   #1
rexxi
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Location: Santiago, Chile

Join Date: Jun 2012
Posts: 20
Default How to split fastq cointaing shared forward barcodes but different reverse ones?

Hi, I need your help because I'm completely lost with that. I received a paired-end sequencing containing many samples in a forward and reverse paired end fastq set of files as shown below.
The reason the sequencing center bring us the sequencing in that format is because they used a different set of primers wich allow them to improve the sequencing quality.

Code:
Librerires_S4_L001_R1_001.fastq 
Librerires_S4_L001_R2_001.fastq
I was expecting to find a software that could extract the samples in a way similar as shown below, not because is just a personal plan but because is commonly used in some softwares (qiime is an example).

Code:
##  [1] "PN1R1_L001_R1_001.fastq"   "PN1R1_L001_R2_001.fastq"  
##  [3] "PN3R2_L001_R1_001.fastq"   "PN3R2_L001_R2_001.fastq"
My problem started when I figured out that some of those samples share the forward barcode, but the difference is in the reverse one, and Iīve never seen something like that. I assume is a feature of modern sequencing platforms with high capacities and with the propper sofware those could be easily splitted and assign to propper derived fastq files.

Code:
Sample         Espacer Forward   Espacer Reverse
1   PN1R1             A                B
2   PN3R1             A                C
3   PN1R2             B                C
4   PN3R2             B                D
As you can see, the forward A is contained in two samples, but those doesnīt have the same reverse barcode. As example of the files, show that contain a barcode that can be in the forward and the reverse, an index doing a difference and the forward and reverse primer.

Code:
  SAMPLE              BARCODE        INDEX   SPECIFIC PRIMER
  For_A  FORWARD    CCTAAACTACGG            CCTACGGGNGGCWGCAG
  For_B  FORWARD    TGCAGATCCAAC      T     CCTACGGGNGGCWGCAG
  Rev_B  REVERSE    TGCAGATCCAAC      A     GACTACHVGGGTATCTAATCC 
  Rev_C  REVERSE    CCATCACATAGG      TC    GACTACHVGGGTATCTAATCC 
  Rev_D  REVERSE    GTGGTATGGGAG      CTA   GACTACHVGGGTATCTAATCC
What I need is to find a sofware that could pick the samples acording to their respective barcodes, even if those are shared in some side and separate between samples. I've been trying some softwares (qiime1, fastx, mothur) but nothing worked as expected. Also I wanted to check qiime2 and SeekDeep too, but at this point I donīt want to waste time checking each software without having a real idea of what they can do.

Does somebody know that kind of post processing and give me a tip of a program which does that kind of job? I would be totally grateful for any hint.

Sorry for this large post but I just wanted to give as much details as I could. Thanks for your time
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Old 03-06-2019, 06:08 PM   #2
cement_head
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Default

Did you ask the sequencing centre how they typically demultiplex?
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Old 03-07-2019, 03:26 AM   #3
Bukowski
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It kind of sounds like your sequencing provider hasn't bothered to demux on the dual index.

You might be able to use bbtools Demuxbyname.sh

http://seqanswers.com/forums/showthread.php?t=58221

There's quite a few other threads on the site about it

http://seqanswers.com/forums/showthread.php?t=83723

I'd be going back to the sequencing provider to get them to do the job properly with bcl2fastq.
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Old 03-07-2019, 09:31 AM   #4
rexxi
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Location: Santiago, Chile

Join Date: Jun 2012
Posts: 20
Default

Hi! thanks for your reply. Actually I asked the reason why they didinīt performed a demultiplex propperly and they explained me that they used a protocol to improve quality and supposedly bcl2fastq would not work as it should.

in the other hand I received a sofware called fqgrep which should solve that problem. At the moment I've just compiled it and I will do the first tests. If it works I will post the solution. If it doesnīt I will try the suggestion posted by Bukowski.

Thanks for your time!
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