Trimmomatic Help Single Reads Error

[Hilary April Smith]

Hi. I just came across/downloaded the trimmomatic program, but in trying to use it to trim my single reads I am obtaining the errors below. I created a fasta file with my adapters (named as >Universal etc., with the full TruSeq adapter sequences). It seems the program is reading the adapter sequences and starts, but then I receive the errors about the fastq header. I can change the fastq line if I know what it should look like, but I can't discern the format required. (As it is I've had to do some manual adjustments; the sequencing facility gave me separate fastq files for the barcode/index and seq. read files ... without modification my fastq name actually ends in 2:N:0. Note also the last sequence is just my addition of a partial adapter read to make sure Trimmomatic kills it). My short test input file, and the error message, are below. Any help is greatly appreciated!!!


@JLK5VL1:2351BE1ACXX:6:1101:1465:2207 1:N:0:CTTGTA
CTTGTAAGCTGGCTTTAATCGCATCGCTTGAACTTTTTCGCCGACCAGACACACAAATGAATACAGCCCCCTCCGTTTTGACTTGCGCCCCCATCAGAGAGACCAACA
+
@@@DD;DBC@FFFFFHFFHHGJJJJJJFHHGCFHJJJJFFIIJJJBH(BCHEGHEHHEEFE;CEF>@A=?B?BD=ACDB?<ACDCC>BBDDDBCDDCDD@<??<CDD@
@JLK5VL1:2351BE1ACXX:6:1101:1518:2135 1:N:0:CTTGTA
CTTGTAATTTTTTGGTGCGTTTGTTTTTCACAACTGTGTTTTCATATTTCCCTATTTCCCTTGTTCTATTTCCGTTCGACTAGCGCACAGGTAGAAAGTGTTATTTTC
+
@BCFFADBCCFFFDFFHHHHGGIGIIIHIIIJIIIICEGEHIGIIGIIJIIJIGIIHDGIGHHFGHHJJIJG@EHHFEFDDCEDDBDD@?A>:ACCA>>AC@CCCD@:
@JLK5VL1:2351BE1ACXX:6:1101:1676:2243 1:N:0:CTTGTA
CTTGTAACTGTAACCAACTACTACCATTCGCAAGCAATTTCGAGCGAGATGGGTGTATACTTAATACAGACAGACCGTGAGTTGGGATCACACGGCGATGAGCATTCA
+
??1=?4:?@@AAB>B<FDFBFGFG?<FHIIDBGEGC3CFFD?CFIAE<DADFB8;5=F>;C)=4=@CFEEF@3?BDC>??@A@BB@@BBBB>><<9>>5-::>@AABA
@JLK5VL1:2351BE1ACXX:6:1101:1767:2179 1:N:0:CTTGTA
CTTGTAACAACGATCGTATCCTTCGACCGAAACGGTTTAAGCTTTGCTGAAGTAGGTTTTACCCACAGGTTACAGCTGCACTACGCCAACGAACGAAACACTCGACTA
+
@B<DBDD@@@FDDFFFHHGHIIIII<FIGGGEIIIEHHCHBBFGGIIDGFEG@@E@CDHEAAEEEFFFD>CEEEDDDDDDCCCC?:@DDDB<BB<BDB>C@(>?2529
@JLK5VL1:2351BE1ACXX:6:1101:2314:2166 1:N:0:CTTGTA
CTTGTAAGATAAGTGCACGGCAATTGCTAGTTTACCGCTGAATATCGCCGCCCGGATCATTGAGTTCAACGGGTTTGTACCCCTAGGCAGTTTCACGTACTATTTGAC
+
@B@DDDDBCCFFFDFHHHHHJJJJJJJJIJIJJJJJJJJJJIIGIIJJJFIJJJHEFFFEECCCEDDDEDDDDDDDDDEEDDDDDDDDDDDCEDEDBBDDD<CDECD@
@JLK5VL1:2351BE1ACXX:6:1101:2345:2192 1:N:0:CTTGTA
CTTGTAACTGGCACAGATCTTGGTGGTAGTAGCAAATATTCGAACGAGCTCTTGGATGACTGAAGTGGAGAAGGGTTTCGTGTCAACAGCAGTTGAACACGAGTTAGC
+
BC@FFFFCCCFFFFFHHHHHJJJEGIHIJHJJIJJJJIIIIIIJJJGIJJJJJJJFEGGIJDCGIFFIBHGHJHH=ABDDECDEEEDDDDCBDDCDDCDDBBDB(:@C
@JLK5VL1:2351BE1ACXX:6:1101:2311:1710 1:N:0:CTTGTA
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG
+
BC@FFFCCCFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

$ java -classpath trimmomatic-0.22.jar org.usadellab.trimmomatic.TrimmomaticSE -phred33 -trimlog trimlogfile.txt head_rnaseq3_BC11.fastq test1.fastq ILLUMINACLIP:Adapters.fasta:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
TrimmomaticSE: Started with arguments: -phred33 -trimlog trimlogfile.txt head_rnaseq3_BC11.fastq test1.fastq ILLUMINACLIP:Adapters.fasta:2:40:15 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Using Clipping Sequence: 'CTAGCCTT ##... (it goes on for all 36 adapter lines, where I gave it sequences for each adapter in the forward, reverse, complement, and rev complement direction. This part looks fine, but then):

ILLUMINACLIP: Using 0 prefix pairs, 36 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Exception in thread "main" java.lang.RuntimeException: Invalid FASTQ name line:
at org.usadellab.trimmomatic.fastq.FastqParser.parseOne(FastqParser.java:51)
at org.usadellab.trimmomatic.fastq.FastqParser.next(FastqParser.java:105)
at org.usadellab.trimmomatic.TrimmomaticSE.processSingleThreaded(TrimmomaticSE.java:55)
at org.usadellab.trimmomatic.TrimmomaticSE.process(TrimmomaticSE.java:197)
at org.usadellab.trimmomatic.TrimmomaticSE.main(TrimmomaticSE.java:262)

[Hilary April Smith]

Hi. Nevermind; I think I may have fixed this...

[tonybolger]

At a guess, i'd say you have a blank line somewhere in your file, quite likely an extra black line at the end.

If you do a line count using wc -l, you should see a count which is evenly divisible by 4.

[Hilary April Smith]

Yes. I am running it now on my full fastq files and from what I've seen so far, it seems to be working great! It's fantastic to have such an efficient tool for trimming adapters and quality filtering. Thank you.