[smitra]

Yes it is tab separated ... I just copied and pasted here...thus looks like so

[GenoMax]

Is your barcode file tab separated format? Hard to tell from the post.

[smitra]

And also the same is happening if I replace the first C with an N in barcode file

#SampleID BarcodeSequence
AP1E NGTAACCA
AP25E NGTACCCA
AP5D NGTAAGAA
AP8C NGTAGATA
P29F NGTAGGCT
P30N NGTATTCA
P31B NGTCAAGA
P35C NGTATTTC
V2A NGTCCAGG
V3J NGTCACAG

N85567:testdata mitras$ cat test_out_R1.fastq | fastx_barcode_splitter.pl --bcfile mapping2_bcfile_new.txt --prefix /Volumes/Cristina/Mr.DNA_2016/fastq_files/testdata/ --bol --mismatches 0
Barcode Count Location
unmatched 1000 /Volumes/Cristina/Mr.DNA_2016/fastq_files/testdata/unmatched
total 1000

[smitra]

Here first I tried to remove all N from the read files as you suggested earlier and also remove C from barcode file:

So now

 mitras$ less test_out_R1.fastq 

@M02542:124:000000000-AKFBJ:1:1101:13841:1000 1:N:0:5
GTACCCAAGGGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACANNCNNGTCGAACGGTAGCNCAGAGAGCTTGCTCTNGGNTGACGAGTGGCGGACGGGNGANTAATGTCTGGGAAACTGCCCGATGGAGGGGGATANCTACTGGANANNGNNGCTAATACCGCATAACGNCGCAAGACCAAAGAGGGNGANNTCAGGGCCTCTTGNCATCGGATGNNCCCAGATGGGATNGGCTTGTAGGTGAGGTAAGNGCTCACGCNGGCGACGATCCCTAGCTTGGNNGNGAGG
+
8ABCFGGGGGGGGEEGGGGGGGG<FGGGFFGFGFGFGGEG@FGEEGGCFGGGGG?##:##6:CFFGGGDG<CG#:CCFFGEGGGGFAFG#:<#:BBFF7FFGDGGGGGGGD#8+#+:BFGGGGGGGCFFGDGG<FGGGECCGDEGGGF@#611:D,>>#6##6##66<1CF@7FFFGEGF7E#41=8=EGFFG7*?CF>>#22##2*2;@;8C8CFC<#/2AC=E*:5##/2:CFCG+8**+#*1*1552<+*+0+8D6D4+#1**)**)*#*15/*//7>5:5<.*,*)0)##1#..73
@M02542:124:000000000-AKFBJ:1:1101:12174:1002 1:N:0:5
GTAACCAAGGGTTTGATCCTGGCTCAGGATGAACGCTAGCTACAGGCTTAACACANNCNNGTCGAGGGGCAGCATTTCAGTTTGCTTGCNAANTGGAGATGGCGACCGGCGNACNGGTGAGTAACACGTATCCAACCTGCCGATAACTCNGGGATAGCNTNNCNNAAGAAAGATTGATACCCNATGGTATAATCAGACCGNATGGTCTTATTATTAAANAATTTCGGTNNTCGATGGGGATGNGTTCCATTAGGCAGTTGGTGTGTTAATGNCGCACCAAACCTTCCTGTGANNGNGTTT
+
8ACCGGGGGGGCFGGGGGGGGGGGGGGGGGGGFGGGGDGGGGGGGGGFGGGGGGG##:##6:CFGFDEGGGGDGGGFGGGFGGGGGGGG#:C#66=,CFFFGGGG@FGEE7#++#:BBFFGGGFCFGGGGGGCGDGGGFGGGGGGGGC=#8@<<<FGG#5##8##86DCF<FCCC:BFCFFF#6>F>FGG92;@CFFGF@#116*=CF<CG?@CFFFG#3;5375:CG##212**<5C5/::#11:91A>+<>C6CE<FC:*****0:FB<#1*)//75<F30762*-2)**##1#0)0.

And the barcode file:

#SampleID BarcodeSequence
AP1E GTAACCA
AP25E GTACCCA
AP5D GTAAGAA
AP8C GTAGATA
P29F GTAGGCT
P30N GTATTCA
P31B GTCAAGA
P35C GTATTTC
V2A GTCCAGG
V3J GTCACAG

Still I am having the same problem :

N85567:testdata mitras$ cat test_out_R1.fastq | fastx_barcode_splitter.pl --bcfile mapping2_bcfile_new.txt --prefix /Volumes/Cristina/Mr.DNA_2016/fastq_files/testdata/ --bol --mismatches 0
Barcode Count Location
unmatched 1000 /Volumes/Cristina/Mr.DNA_2016/fastq_files/testdata/unmatched
total 1000

[GenoMax]

Can you try replacing the first C with an N in your barcode file and see if fastx-splitter would accept that as a valid pattern and do demultiplexing?

If that does not work then you could use bbduk from bbmap suite (with forcetrimleft=1) option or HEADCROP:1 option for trimmomatic to remove that first base (which is N) from all reads.

[smitra]

Thanks ... but how can I do so..any quick way ? Do you suggest to use fastx_trimmer?

[GenoMax]

I suppose you could do two things. Remove the 1st base (if it is always N, which is kind of odd, see below) from all reads and remove the first C from your barcode file.

Hypothesis: Reason that first base is an N is because every sequence in this case will actually start with C (and then have GT). I am surprised that this worked for 2nd base onwards. Having low nucleotide diversity like this is not recommended.

[smitra]

Thanks for replying GenoMax

#SampleID	BarcodeSequence
AP1E	CGTAACCA
AP25E	CGTACCCA
AP5D	CGTAAGAA
AP8C	CGTAGATA
P29F	CGTAGGCT
P30N	CGTATTCA
P31B	CGTCAAGA
P35C	CGTATTTC
V2A	CGTCCAGG
V3J	CGTCACAG

But as the fastq files look like (I assume the bold red part is the barcode with one N)

mitras$ less test_R1.fastq

@M02542:124:000000000-AKFBJ:1:1101:13841:1000 1:N:0:5

NGTACCCAAGGGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACANNCNNGTCGAACGGTAGCNCAGAGAGCTTGCTCTNGGNTGACGAGTGGCGGACGGGNGANTAATGTCTGGGAAACTGCCCGATGGAGGGGGATANCTACTGGANANNGNNGCTAATACCGCATAACGNCGCAAGACCAAAGAGGGNGANNTCAGGGCCTCTTGNCATCGGATGNNCCCAGATGGGATNGGCTTGTAGGTGAGGTAAGNGCTCACGCNGGCGACGATCCCTAGCTTGGNNGNGAGG

+

#8ABCFGGGGGGGGEEGGGGGGGG<FGGGFFGFGFGFGGEG@FGEEGGCFGGGGG?##:##6:CFFGGGDG<CG#:CCFFGEGGGGFAFG#:<#:BBFF7FFGDGGGGGGGD#8+#+:BFGGGGGGGCFFGDGG<FGGGECCGDEGGGF@#611:D,>>#6##6##66<1CF@7FFFGEGF7E#41=8=EGFFG7*?CF>>#22##2*2;@;8C8CFC<#/2AC=E*:5##/2:CFCG+8**+#*1*1552<+*+0+8D6D4+#1**)**)*#*15/*//7>5:5<.*,*)0)##1#..73

@M02542:124:000000000-AKFBJ:1:1101:12174:1002 1:N:0:5

NGTAACCAAGGGTTTGATCCTGGCTCAGGATGAACGCTAGCTACAGGCTTAACACANNCNNGTCGAGGGGCAGCATTTCAGTTTGCTTGCNAANTGGAGATGGCGACCGGCGNACNGGTGAGTAACACGTATCCAACCTGCCGATAACTCNGGGATAGCNTNNCNNAAGAAAGATTGATACCCNATGGTATAATCAGACCGNATGGTCTTATTATTAAANAATTTCGGTNNTCGATGGGGATGNGTTCCATTAGGCAGTTGGTGTGTTAATGNCGCACCAAACCTTCCTGTGANNGNGTTT

+

#8ACCGGGGGGGCFGGGGGGGGGGGGGGGGGGGFGGGGDGGGGGGGGGFGGGGGGG##:##6:CFGFDEGGGGDGGGFGGGFGGGGGGGG#:C#66=,CFFFGGGG@FGEE7#++#:BBFFGGGFCFGGGGGGCGDGGGFGGGGGGGGC=#8@<<<FGG#5##8##86DCF<FCCC:BFCFFF#6>F>FGG92;@CFFGF@#116*=CF<CG?@CFFFG#3;5375:CG##212**<5C5/::#11:91A>+<>C6CE<FC:*****0:FB<#1*)//75<F30762*-2)**##1#0)0.

But as you can see I have N, so may be I need to allow 1 mismatch for the barcode.
Thus I tried code as:

cat test_R1.fastq | fastx_barcode_splitter.pl --bcfile mapping2_bcfile.txt --prefix /Volumes/Cristina/Mr.DNA_2016/fastq_files/testdata/ --bol --mismatches 1

Thanks for helping
smitra

[GenoMax]

Can post a few lines of your fastq file and the mapping file?

[smitra]

Hi Everybody,
I came back to this thread again as I am getting a very similar problem to the first post by janderson.

My code works fine:

cat test_R1.fastq | fastx_barcode_splitter.pl --bcfile mapping2_bcfile.txt --prefix /Volumes/Cristina/Mr.DNA_2016/fastq_files/testdata/ --bol --mismatches 1

But none of the output files contain any reads except for the mismatched file.

This data we got from Mr.DNA and raw fastq file for 10 sample together which I need to split. Johnathon's later suggestion din't help.
Can anybody help please?
Thanks,
smitra

[smitra]

Hi vivi7,
I guess you need to provide your fastq or fasta file. You haven't provide that.
Use as

cat File.fastq | /usr/local/bin/fastx_barcode_splitter.pl --bcfile mybarcodes.txt ...other options if you want.

[vivi7]

fastx_barcodes_splitter issue with run

Hi,

I saw the post and I hope maybe some of you can help me

When I run fastx_barcode_splitter.pl with this script

/usr/local/bin/fastx_barcode_splitter.pl --bcfile ./Barcodes9nt.txt --prefix ./Rescued9nt --suffix .fq –bol

In the command line it looks like is running (no error message, no > sign), see attachment for screenshot.
However is not running at all, I can see with top that is not using any memory or CPUs and it has been ‘running’ for days on a very small file without producing any results.
The input file is in the STDIN folder as supposed to.

I would be very grateful if you could suggest what might be wrong.
Thanks in advance
Vivi

[carmeyeii]

Thank you, chadn!

Of course this was the easiest solution.

The barcode is:

REVERSEPRIMER	[COLOR="red"]CGCGTCAGCATTGTTCATAC[/COLOR]

Read 1 begins with a perfect match to the barcode.

@HWI-M00149:16:000000000-A12VK:1:2114:17873:29127 2:N:0:
[COLOR="Red"]CGCGTCAGCATTGTTCATACAAAGCTAC[/COLOR]TTAGTTGCTACGAAGCAATACATTGTTAGTTGTTAACTACTCCCCCCTCTTGTTTTNNNCNNTNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNN

Read 2 has an extra base at the beginning, followed by a perfect match to the barcode.

@HWI-M00149:16:000000000-A12VK:1:2114:17873:29128 2:N:0:
A[COLOR="red"]CGCGTCAGCATTGTTCATAC[/COLOR]AAAGCTACTTAGTTGCTACGAAGCAATACATTGTTAGTTGTTAACTACTCCCCCCTCTTGTTTTNNNCNNTNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNN

Read 3 is missing the first base of the barcode.

@HWI-M00149:16:000000000-A12VK:1:2114:17873:29129 2:N:0:
[COLOR="red"]GCGTCAGCATTGTTCATAC[/COLOR]AAAGCTACTTAGTTGCTACGAAGCAATACATTGTTAGTTGTTAACTACTCCCCCCTCTTGTTTTNNNCNNTNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNN

By trimming the first base of every read,

we are left with

Read 1 [now missing 1 base at the beginning]

[COLOR="Red"]GCGTCAGCATTGTTCATACAAAGCTAC[/COLOR]TTAGTTGCTACGAAGCAATACATTGTTAGTTGTTAACTACTCCCCCCTCTTGTTTTNNNCNNTNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNN

Read 2 [now perfect match]

[COLOR="red"]CGCGTCAGCATTGTTCATAC[/COLOR]AAAGCTACTTAGTTGCTACGAAGCAATACATTGTTAGTTGTTAACTACTCCCCCCTCTTGTTTTNNNCNNTNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNN

Read 3 [now missing 2 bases at the beginning]

[COLOR="red"]CGTCAGCATTGTTCATAC[/COLOR]AAAGCTACTTAGTTGCTACGAAGCAATACATTGTTAGTTGTTAACTACTCCCCCCTCTTGTTTTNNNCNNTNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNNNNNNNNNNNNNNN

and by using

--mismatch [COLOR="red"]4[/COLOR] --partial [COLOR="red"]4[/COLOR]

all reads will be matched to the barcode.

The --4 doesn't make sense to me, as I thought this would be --2, but this is the only thing hat gets it to work, so...

Thanks a lot!

Carmen

[chadn737]

A quick and dirty solution would be to trim of the first base pair of all your reads and then just use FastX barcode splitter with --partial

[carmeyeii]

Hi everyone,

I've been using the FastX Barcode Splitter successfully, but regarding the --partial option, I have realized I'm losing some reads with a particular problem:

With --partial 1

The barcode

CGCGTCAGCATTGTTCATAC

will pick up the read

[COLOR="purple"]GCGTCAGCATTGTTCATAC[/COLOR]AAAGCTACTTAGTTGCTACGAAGCAATACATTGTTAGTTGTTAACTACT

since it is missing just one base at the left end to match the barcode exactly.

However, the read:

C[COLOR="Purple"]CGCGTCAGCATTGTTCATAC[/COLOR]AAAGCTACTTAGTTGCTACGAAGCAATACATTGTTAGTTGTTAACTACT

will not be taken as matching the barcode, since it has one extra base at the beginning. Unfortunately, there are many reads that fall into this category, but not all of them begin with the extra 'G'.

Do you use anything else to get around this?

Thanks!
Carmen