Trouble with FASTQ joiner

Question: Trouble with FASTQ joiner

18 months ago by

nday • 0

United States

nday • 0 wrote:

Hi Biostars community, I attempting to run some initial QC and manipulation tools on some rnaseq data (illumina 1.8). I have my trimmed data from Trim Galore!, however I am unable to pair the trimmed data with FASTQ joiner. Whenever I take the two paired outputs (in fastqsanger format) from Trim Galore and use them in FASTQ joiner, the program will run, but returns a message states that 0% of reads were paired, no matter the "old" or "new" header option is selected.

"There were 22817078 known sequence reads not utilized. Joined 0 of 22817078 read pairs (0.00%)."

My Trim Galore data is in the following format: @D00472:148:CB1Y1ANXX:2:2201:1059:2134 1:N:0:ATCACG CTCAGGCATAGGTCACCAGCTTTCGGGTCGTTTGCCAACTGCTCAACCTCTGCACAGTCACAAGTGACACGCACAGGGCCGTGGTGCGCTGCACTCCG + BBBBBBFBF/</bbffff>

Am I missing something?

I have used this workflow in the past with success, but now it does not seem to work. I have noticed that this error popped up about 5 years ago, however the work around solution calls on a tool that is not available anymore. Any help/suggestions would be greatly appreciated.

Thanks.

Nick

rna-seq galaxy • 491 views

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modified 18 months ago • written 18 months ago by nday • 0

18 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Are you certain that the two fastq inputs are a pair? The headers must match between the two, either a "1 + 2" or "1 + 3" pairing. The sort order must also be the same between the two inputs for the tool to join them.

If you are not sure, you can post the first 20 lines of each dataset (or any multiple of 4 lines, so that at least 5 full sequences are shown) and we can help with troubleshooting. Try pasting the sequence data into a gist or another online site that will preserve format and share the link - or - you can paste back directly in a comment here and use the "code sample" text formatting option.

Thanks! Jen, Galaxy team

ADD COMMENT • link written 18 months ago by Jennifer Hillman Jackson ♦ 25k

18 months ago by

nday • 0

United States

nday • 0 wrote:

Hi Jen, Below is a sample of the two fastq inputs (from Trim Galore!) that I am using for FASTQ Joiner. (They have been through FASTQ groomer previous to Trim Galore!). Taking a closer look at the sequence identifiers of my forward and reverse files, I notice that the flowcell lane and tile number within the flowcell lane is different between the files. Could this affect the program's ability to pair reads if the identifiers do not match up in the Coordinates section? Old data from a different experiment has matching coords but different flags for forward and reverse reads, but here, it seems to be the opposite. I am also certain that these files are pairs based on the file names that were assigned to them from the sequencing facility.

Forward:

   @D00472:148:CB1Y1ANXX:1:1102:1172:2173 1:N:0:CGATGT
GTGGGTTGGTAGCTCCGAGCATGGAACGTCCTTGCTTGACGACGTCAAGTCCTTGCCAGACCATGGCGACGACTGGTCCAGAGCGCATGTACTCGATG
+
BB<B/FFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFF</BBFFF<FFFFFFFBBBFFFFFFFFFFFFFFFFFFFFFFFFFF/FBFFFFFFFFFFF
@D00472:148:CB1Y1ANXX:1:1102:1195:2206 1:N:0:CGATGT
CTCAGCGCGCAAATGATCGATACAATCATACACGTCATTTTCATCTTTTCCTGCAACGACAACCAAGTTTGGATCTTCTGCTCCTTGACGTGGAAGACG
+
BBBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@D00472:148:CB1Y1ANXX:1:1102:1068:2216 1:N:0:CGATGT
CTGGGACGGCCTCTGGAAGAGATTCGTGATGCATCTCAACGGACTTGACTTCAGTGGTGACGTTTTGTGGAGCGAAGGTAACGACCATTCCTGGCTTGAT
+
BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@D00472:148:CB1Y1ANXX:1:1102:1217:2223 1:N:0:CGATGT
CACAGTGCAAAAAGTAGTTCACCCAGTGAGGATCGTCGTAGTGAGCCTCGACTTGTGCAATTTCATTATCGTGATGCGGGAACTTCAGATCGAAACCGCC
+
BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFF
@D00472:148:CB1Y1ANXX:1:1102:1480:2144 1:N:0:CGATGT
CACAGTTTGAAGCGCGGGGGCGGGGTCCTTCAAGAACATGATGGTATTGATGAAATAGCTTCGCAAGGGGTAATTAGCGGGGGATAGGAAAGGGGAAA
+
BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

Reverse:

    @D00472:148:CB1Y1ANXX:2:2201:1190:2118 1:N:0:CGATGT
CTCTAGCATTTGAGTTTAGTTCCAAAGGAAGCCGTACAAAATCAAAAATCACCGGGGAATTCTACTACTCGATGGTAGTATTATTCACGGGTCGAACGTT
+
/<<BBF/<FFFBBFFFFF/BB<FF/<FFFFBFB<<FFFFF<<<FFFBFFFFFBFBF///<FBFFFFBFFFFFFFBFFFFBFFFFFBFFFBFB/B</FBFB
@D00472:148:CB1Y1ANXX:2:2201:1128:2160 1:N:0:CGATGT
CCTCTTTCTCCCCCACCGAGTTTTCTCCACGAGATGCTTATATCCTCGTCAACGATGCCACAGTTTTAGTCTCATCCACTAGAAAACATCATTTTTTCG
+
BBBBBFFFFF<//</FFF/<FFFFFFFBFFFFFFFFFFFFFFFFFFFFF/<<FFFF/7BFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFF/7
@D00472:148:CB1Y1ANXX:2:2201:1342:2132 1:N:0:CGATGT
GGAAAAATCGCAATTTTGGGTAAACGATATACATTGAAATCATTTTTAAATATTGGCAATATTATTCAGAAGCTTATCGCCAGTTATCAATGCACTTGT
+
BBBBBBFFFFF/<FFFFF/<<F//B<BFFFFFFFFFFFFFFFFFFFFFFFFFFFF</<FFFFFBFFBB<FFFFFFFFFF/FFFFFFFFFFFFFFFFFBF
@D00472:148:CB1Y1ANXX:2:2201:1327:2143 1:N:0:CGATGT
GTTTGAAGATCAGACACCGCAGCCTACGTTCTTTGTTTTTATGAAGAATTTAGACGATGGAGAGGACTTCTCCCCCCACACACACAAAAAAAAACAAA
+
BBBBBFFFFFB/B<FBFF/FFFFFFF<FFFFFFFFFFFFFFFBFFFFFFFFFFBFB/<<FFFBFFFFFFFFFFFFFFFFFFFBB<BFBFFFF<FBFFF
@D00472:148:CB1Y1ANXX:2:2201:1420:2191 1:N:0:CGATGT
CTGGTTGATGAAAGTCAAGAGTCTGGCGATGTTCTTGCGGACAACGCGAATCTTGGGGAGCTTAGAGGCAGCTCCTCCAGTGACCTTCGAGACACGG
+
BBBBBFFFFFF/BFFFFF/BFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF/<BFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFF

ADD COMMENT • link modified 18 months ago • written 18 months ago by nday • 0

I agree that the problem is with the sequence labels. Before going forward, asking for a clarification from the sequence provider would be a good idea. Samples can get mixed up.

ADD REPLY • link written 18 months ago by Jennifer Hillman Jackson ♦ 25k

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