deML: ERROR: Discrepancy between fastq files, different names with first index

Question

[boonfei]

Dear all,

While trying to find a program to demultiplex dual index illumina paired end file, I came across the program deML which I believe can be very useful.

While running the program, i received this error message: "ERROR: Discrepancy between fastq files, different names with first index @M04129:63:000000000-D656Y:1:1101:15821:1334 and @F1"

The multiplexed file contain "hidden index" in the sequence which are not shown in the sequence header. Do you think whether the program is trying to look for the barcode sequence in the sequence header? This will not work as the barcode sequence on the sequence header is actually not the same in the fastq file required by deML to demultiplex.

Any help with this is much appreciated.

[Felipe]

Hi Gabriel R., I have the same kind of issue trying to run deML with paired-end reads using iTrue indexes, and I am unsure if I understand what you mean by having the same name. Do you mean the name of the indexes should be the same as the reads in my multiplexed file?

Thanks,

[Gabriel R.]

Hi Felipe, can you please have a look at the fastq files in the test data? The headers for the reads+indices are the same save for the /1 and /2 in the forward and reverse reads:

 zcat testData/todemultiplex.fq1.gz |head
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15604:1334/1
 TTCTTCCTTCCTTCTTCTCTTCCTCCTTTCTTTCCCTCCTTTCCTTTCTTTCTCCTTCTTTCCTCCCCTTTCCTTCCCCCTTTCTTTCTCTCCCTCTCTCCTCCCCCTTCCTCCTCGCCTCCCTCCTCCCCCTCCTCCCCCCCCCTCCCT
 +
 9<EDEDCBCHHD>GIC<B.EFHGCI;@EH@I>C0A-DECCDCAII:B<BFIG:GGDE8G:C;@A8G3:IC@IEFFD8<I1I@DHCD5IE<C8IIFI6IGDGE4FH;CDEIIFHIFI8;IE?GIEHHEIIBFB7I(EI/IH4IC3:=94?.
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15289:1339/1
 TTCTTCCTTCCTTCTTCTCTTCCTCTTTTCTTTCTTTCCTTTCCTCCTCTTCCCCTTCTTCCCCCCCCTTCCCTTCCCCCTTTCTCTCTCCCCCTCCCTCCTCCCCCTTCCTCCTCCCCTCCCCCCTCCCCCCCTTCCCCCTCCTCCCCC
 +
 9:ECCC4BDHGC7GIC@ACDEHGCICCEGDH:B3ECD>GCDCFIIFFCIEHG8GHDE6GDI8GF<GD2H=BIEEEG2=IHIC>HDD:IDAH?IIEHDIF<GECDHA0DEIIDHIEHGFIDFGIGHHEIIGFH?IDEII4:F3C@C@45>I
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15894:1346/1
 TTCTTCCTTCCTTCTTTTTTTCCTCTTTTCTTCCTCTCCTTTCCTTCCTTTCTCCTTCTCTCCTCCCCTTTCCTTCCCCCTTTCTTTCTCTTCCTCCCTCCTCCCCCTTCCTCCTCTCCTCCCTCCTCCCCCTCATCCCCCCCCTTCCCT

 zcat testData/todemultiplex.fq2.gz |head
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15604:1334/2
 TTTTTCTTTTCTTCTCCTTCTCCTTCTCTTTTCCTTTCTCTTCTTTCCCTTCTCCCTCCCTCCTTTCCCTTCCCTCTTCCCTTCTTCTTTCTTTTTCTTCCTCCTTTTTCCTTTTTTCTCTCTTTGTCCTTCTTTTTCTTTTCCTCTCGC
 +
 9<@D;4@DAFCAB.E8EC9IFI=DD4D1IEHEGGIEDIEIEFIGIDIGIFHHDHIHHG>4DGGGBFGIBFGGGIEDI=IB@EE@ED?DHHEIHCEDHDFHFFGIFEED;>GDCDDE8GEIDGDDE4D1HEDGFE28DG<E5BC@CB72/I
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15289:1339/2
 TTCTCCTTCTCTCCTCCTTCTCTTTCTTTTTTCCTTTCTCTTCTTTCCCTTCCCCCTCCTTCCTTTCCTTTCCCTCTTCCCTTCTTTTTTCTTTCTTTTCCTTCTTTTTCTTTTTTTTCTCCTTTCTTCTCTCCTCCCTTCTTCTTCCTT
 +
 9=HC8E@BEDHB$7EGFCDIFICDDGDBIEGEGGIEDIEIEGIFIEIHIFHHIIIHGIGDADGGCFGIEEEGGIEDIBICDEEHEDCDGGFIIDGDD;FHEEDIFDE>EHBEEDFED@GDHGC>E@DFIEF6GFCG@FDEGCA@:>3/@.
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15894:1346/2
 TTCTTCCTTTCTTCTTCTTCTCCTTCTTTTTTCCTTTCTCTTCTTTCCCTTCTCCCTCTCTCCTCTCCTTTCCCTCTTCCCTTCTTTCTTCTTCTTCCTCCTCCTTTTTCCTCTTTTCTCCCTTTCTCCTTTTTCTTCTTCTCCTCTCCC

 zcat testData/todemultiplex.i1.gz |head
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15604:1334
 CCCTCCT
 +
 @IID@II
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15289:1339
 CCTTTTT
 +
 DFEAIFI
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15894:1346
 CCTCCTG

 zcat testData/todemultiplex.i2.gz |head
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15604:1334
 TCGCCGG
 +
 BIFIIEF
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15289:1339
 TTCTCCG
 +
 DCIFIIG
 @M00518_0198_000000000-A5KVK_VW_17012014:1:1101:15894:1346
 CCCGCTG

Answer 1

[Gabriel R.]

This indicates that the defline for the forward read and the first index are not the same. You have a record with a defline "@F1". Are you sure this is the per read index? Could you perhaps post the first 40 lines of both files?

[boonfei]

This is the F-index file

@F1
ATCACG
+
DDDDDD
@F2
CGATGT
+
DDDDDD

This is the reverse index file:

@R9
GATCAG
+
DDDDDD
@R10
TAGCTT
+
DDDDDD
@R11
GGCTAC
+
DDDDDD

[boonfei]

Actually I have amended the fastq files to ensure the names are the same but it still doesnt work:

Forward

@F12
GATCAG
+
DDDDDD
@F2
TAGCTT
+
DDDDDD
@F3
GGCTAC
+
DDDDDD

Reverse

@F12
ATCACG
+
DDDDDD
@F2
CGATGT
+
DDDDDD
@F3
CGATGT
+
DDDDDD

[Gabriel R.]

They should have the same names as your forward reads, in your case: "M04129:63:000000000-D656Y:1:1101:15821:1334"