Splitting NuGen barcodes from paired-end sequences

[ester]

Hi all,

Does anybody know a software to split Nugen barcodes that supports PAIRED-END reads?

Thanks,

Ester

[axgraf]

Hi Ester,
I wrote a small program for that issue.

The tool filters the reads by searching for the barcode only in the first read. If found the barcode is removed and written to the output. Note that the input files must be in order.
Small example:

java -Xmx4g -jar DemultiplexNUGEN.jar -i1 laneX_1.fastq laneY_1.fastq ... -i2 laneX_2.fastq laneY_2.fastq ... -b ATTG -o1 ATTG_demulitplex_1.fastq -o2 ATTG_demultiplex_2.fastq -s

Hope I could help. Please keep me informed if it works.

Alex

[ester]

Splitting NuGen barcodes from paired-end sequences

Hi Alex,

Thanks for your help.

I tried to run your program with the following command:

java -Xmx4g -jar DemultiplexNUGEN.jar -i1 s_7_1_sequence.txt -i2 s_7_2_sequence.txt -b ACCC -o1 test.1
-o2 test.2 -s

and got the following error:

de.genzentrum.lafuga.NotFastqFormatException: Read1 has not the same identifier as read2
at de.genzentrum.lafuga.trimmer.Demultiplex.iterateFastqPairedEnd(Demultiplex.java:96)
at de.genzentrum.lafuga.main.MainPairedEnd.main(MainPairedEnd.java:70)

The input files:

>head s_7_1_sequence.txt
@HWI-ST611_0176:7:1:1226:2054#0/1
NGTACTCGTCCACGTCGTTCTCAGAGAGAATATTCTCTCTCCACACATCAGCAGTTAAGGAGGATGTGAAGACAATCTTTTCAACACTATCGGTCTGAGC
+HWI-ST611_0176:7:1:1226:2054#0/1
BYWYW[ZZZZcccccc_cccccccccccccc_ccccccccccccccccc\ccc_ccc\cccc_\cccccVccac______YUcUc\^^^\^^XZ^[X\\\
@HWI-ST611_0176:7:1:1161:2111#0/1
GAGTAGGCCACGCNTTCACGGTTCGTATTCGTGCTGGAAATCAGAATCAAACGAGCTTTTACCCTTTTGTTCCACACGAGATTTCTGTTCTCGTTGAGCT
+HWI-ST611_0176:7:1:1161:2111#0/1
gggeggggggcccBccccccggggfdgeggdbdddgfgfgdgggggeefgegeggbeegedea[gfedaagZeed]]bb`eedfegXgggabaddYaeca
@HWI-ST611_0176:7:1:1197:2111#0/1
GAGCCGCCCGCTCTCTGCTTTCCAAGCCTTTGCGATCTGCTTAAGCAGCTTTGACACCAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTC
arkady Melon_2011/data> head s_7_2_sequence.txt
@HWI-ST611_0176:7:1:1226:2054#0/2
CAAATGGTGGATTTGGAGGTTAGAGGAACAATTAATGTCGTCGAGGCTTGTGCTCAGACCGATAGTGTTGAAAAGATTGTCTTCACATCCTCCTTAACTG
+HWI-ST611_0176:7:1:1226:2054#0/2
gggggggegggggggggggggggdgggggggggggggggggggggggge^cd`cddfeeffbe`d`dddd]eee_XddacaddW[aca`cadcbeMdcbT
@HWI-ST611_0176:7:1:1161:2111#0/2
GGTGGGCCGATCCGGGCGGAAGACATTGTCAGGTGGGGAGTTTGGCTGGGGGCGGCACATCTGTTAAAAGATAACGCAGGTGTTCTAAGATGAGCTCAAC
+HWI-ST611_0176:7:1:1161:2111#0/2
fhdgbgggddfefffegfggfbggggddegeea^eedd^deeebecee^cadUXd\TV]`a[]bdfeeda\VadaabcdcK^V\E]U[TY]Ybbbdb[d\
@HWI-ST611_0176:7:1:1197:2111#0/2
GGTGTCAAAGCTGCTTAAGCAGATCGCAAAGGCTTGGAAAGCAGAGAGCGGGCGGCTCAGATCGGAAGGGCGTCGTGTAGGGAAAGAGGGGAGATTTCGG


Can you help with this?

Thanks again,
Ester

[axgraf]

Hi Ester,
The tool compared the identifier of the reads and stopped because the names weren't the same.
I missed the fact, that paired-end reads could have
a "/1" and "/2" at the end of the identifier, which aren't present in our reads.

I changed the code, so that it should work for your files.

Alex

[ester]

Hi Alex,

Still having problems:


java.lang.NullPointerException
at java.io.File.<init>(Unknown Source)
at de.genzentrum.lafuga.trimmer.Demultiplex.iterateFastqPairedEnd(Demultiplex.java:74)
at de.genzentrum.lafuga.main.MainPairedEnd.main(MainPairedEnd.java:70)


Thanks,

Ester

[axgraf]

Have you used the same parameter as in the last post?
It seems to me, that the -o2 switch was not set.

If I use the same parameter and the same sequences as in the last post, I can run it successfully.

If you copy the parameter out of your last post, the "-o2 test.2 -s"
line is missing.

That could have caused the file not found exception.

Otherwise I need the exact parameter which you have used.

Alex

[ester]

Hi Alex,

You are right. It was my mistake.
The program run but the output file is missing the read name after the +:

arkady Melon_2011/data> more test.1
@HWI-ST611_0176:7:1:2764:2469#0/1
AGGAGTCCGGTATTGTTATTTATTGTCACTGCCTCCCCGTGTCAGGATTGGGTAGATCGGAAGAGCGGTTCTGCAGGAATGCCGAGACCGATACCG
+
gggfggggggdggggggggggggggggegeTedcdeggdfgccZegada`ecXabZX_``\`bMYY`aM^\ZX[S^dabXbBBBBBBBBBBBBBBB
@HWI-ST611_0176:7:1:5412:2350#0/1
CCGGGTGACGGAGAATTAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATC
+
gggfg_gegggggegggdggfggggegggggeggaggd\eefcdbdd[edd`ddeX\\aBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB

Thanks again,

Ester

[eslondon]

You can also use novobarcode, part of Novoalign, to split reads in "buckets" based on barcodes.

[axgraf]

You are right.
Sorry for that. This tools was used up to now only here at our institute.
I changed it.
Hope everything is fine now.

Alex

[ester]

Now it works fine.
Thanks a lot,
Ester

[senpeng]

Hi Ester,
The tool compared the identifier of the reads and stopped because the names weren't the same.
I missed the fact, that paired-end reads could have
a "/1" and "/2" at the end of the identifier, which aren't present in our reads.

I changed the code, so that it should work for your files.

Alex

Dear Alex,
we met the similar problem, and our input format is for CASAVA 1.8, a little bit different with the former one (the position of "1" and "2")
our input are as follows:
@HWUSI-EAS174:6:FC:1:1:1153:945 1:Y:0:
GGGAGGTCGAGGCTGTAGTGAGCTGGGATCGTACCATTTCTCTCATTACGAGATCGGAAGAGCGTGGTGTTGGGACTGAGTGTAGATCTCGGTGGGCGGC
+
25+=70.6;1@@;,;A?=?:19)7;*+++5+?=;+.7;<)3>61*?=;:=BD?B@?222=?8+BB###################################
@HWUSI-EAS174:6:FC:1:1:1288:931 1:Y:0:
GAGGTCGGCTTGGAGTCAGAAAGCTCGGGGCATTGTCTCAGGTCTGTTGCTTCCTAGGAGTGTGAACGATGAGGAAGTTCCTGCATCGCTGAGGACTCAG
+
?+@=6;2;@==B;54;=;=+:785+--/77B?B?D#################################################################
@HWUSI-EAS174:6:FC:1:1:1305:938 1:Y:0:
GGGTTCGCTCGGTGAACTGCACGCCCTTTGAAATGTCTCCTCTCGATTTGGGTGTTTTACTTGATTTTTCTTATATCTTACATCTTTTCTTTAGTCTGTC
+
####################################################################################################
@HWUSI-EAS174:6:FC:1:1:1528:951 1:Y:0:
CGCAAGGACAAAAAACCAAATACTGCATGTTCTCAATCATAGGTGGGAATTGAACAATGAGAACACAGGGACACAGGAACACTCAGATCGGAAGAGCGTC
+
IIIHIHBBIGIIIIIIIIHHBIHIIIIEBIGIIIIDGGBGGGGDGGADGEIIIIGDGEGIHFGHI<IHDGE@HHBIFF@FIFBHHIEG@@HEDDEE>B>3
@HWUSI-EAS174:6:FC:1:1:1551:943 1:Y:0:
GGAGGCTGCTTTTAGGCCTACTATGGGTGTTAAATTTTTTACTCTCTCAAACACCGGGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCG
+
ED=D4FEEE?8BB@B4FBFEE4BFD/:0:4B?;8B*45402921;+86=4CCE?EDDB+DA<ACAD@<GB<0><6?>:4??C>1?###############
@HWUSI-EAS174:6:FC:1:1:1588:935 1:Y:0:
CCGTGATAGTTTTTAGGTGTTAGACACCCCACCTTAAGCTTGTACCTGAAAGCTTTATCTCGTTATAAATAATTCACTGTAATTTAGGGGAGGTATGTCC
+
2+85::1:77)::1:=+9=@@32,@=3<;99@@@F=@4B8B?7C:B?CAB=??8E734282B==77241@##############################

Thus when I run the java, it still shows "Read 1 has not the same identifier as read 2", would you pls help me solve that?

Thanks so much