[Brian Bushnell]

I wonder if maybe a polishing program like Quiver could automatically fix the reference for you. That would be the ideal solution. The problem, of course, is that there are misassemblies or major structural variations with respect to the reference. Hmmm...

I think there are 3 main options.

1) Map with the "local" flag. This will soft-clip the part of the read that extends across the misassembly, so it will not result in spurious variations being called. That's certainly the easiest approach.
2) Try polishing the assembly with something like Quiver, to automatically fix these things.
3) Make a new assembly, if this genome differs substantially enough from the reference that this issue is common.

[GenoMax]

I wonder if you should manually put a break (stretch on N's in that position) to force the two ends apart. While that may resolve this "end" of the reads issue not sure if that is how things are in the real genome.

Are you certain your reads (R1/R2) do not overlap? If they could then you could merge them first before alignment.

Are the reads in the image corresponding pairs (R1/R2)?

[babine]

@Genomax: yes that's exactly what I assume from the read profile. And yes the reference is the sequence with yellow background just below the consensus

[GenoMax]

@babine: What do you mean by "not-so-accurate" reference? Is the reference as depicted in the picture wrong (i.e. there are two ends that are joined that should not be together)? Note: I don't use geneious but I assume one of the lines at the top represents the reference (and the other a consensus?)

[babine]

Sure Brian, here it is! A screenshot from Geneious on an alignment obtained with BBMap as

Executing align2.BBMap [in1=177291_Brachy_300_Brachypodium_distachyon_2x_R1.fastq, in2=177291_Brachy_300_Brachypodium_distachyon_2x_R2.fastq, out=Bdi_1H_AK250130_mapped_bis.sam,
ref=.\resources\Brachypodium_distachyon_2x_AH_AK250130.fasta, K=9, build=3, maxindel=30, pairlen=600, minid=0.9, padding=20, overwrite=t]

"A picture is worth a thousand words"

Best

[Brian Bushnell]

Would it be possible to post an image from IGV, or some kind of text diagram? I can't quite visualize what you're describing.

[babine]

Guidance for mapping with BBMap?

Dear Brian, dear users,

I have reads from an exon based sequence capture and I'm trying to map them to not-so-accurate reference sequences. I started to use BBMap because I hoped it would have a feature that I'm looking for.

My problem is that I'm getting misaligned reads, especially in the end of the reads, when two "stacks" of reads are coming in contact although they should not be but my reference sequence artificially bring them close together. While I would gladly inspect the alignments for those regions and introduce gaps in the reference, I can not do it for more than a few loci. So I was wondering if there is not a more clever way by tweaking the parameters.

I tried a bit with minid, k, padding... but without much success.

Do you have any suggestions on what parameter would have the most impact? Any particular strategy?
Or let me know if you think another algorithm would do it...

Any comment is appreciated...

Cheers

[GenoMax]

That offending sequence may be somewhere around #32. Look further down in the file. Otherwise use BBDuk to retrim your data as suggested by Brian to ensure there are no zero length reads.

[Bioinform]

Are you talking about some lines, my fastq file do have + symbol.

This is my fastq file

@DJB77P1:527:H7RDTADXX:1:1101:2830:2226 1:N:0:
AATTTCCGTCACCCTTTTAAGTCCCCCAGGCGGGGGGCTCGACGAGAGCGACAGACCTTGTGTGTAGAAGTTTCAAAATGCTTATGCATCAAGAGACAGTGCCCTGCCCGAAGATATTTACATTCGGTGTGCCTTGGGCGTATA
+
FFHHHHHJJJJJJJJJJJJICHIIJJIJJIJJJJDDBDDDDBDDBBDDDDDDDDDDDDDDCC??BCCDDDACDDEDDDDDDDEDDDDEECDDDDDDCDDCCDDDCBDBDD<@B<ACDEEDEDDEDED?BBDDDC@>CBBDBB@C
@DJB77P1:527:H7RDTADXX:1:1101:2830:2226 3:N:0:
AGCTCGCCTTACGATGCTCGCACACGAAGACCGCAGAATTAACCAATAACTCCCTGCCTCAAGCCTTGGGCGTATACGCCCAAGGCACACCGAATGTAAATATCTTCGGGCAGGGCACTGTCTCTTGATGCATAAGCATTTTGAAACT
+
=DFFFFHHHHHJJJJJIJJJJJJJJJJJGIJJJJJJJJHHHHHHFFEFFDDEEDEDDDDDDDDDDDDCCDDDDDDDEDDDDDDDDDDDDDDDDDDDCDEEEEDDEEEBDDDDDDDDDDDDDDDDDDDDCDDDDDDDCDDDDECDDDDD
@DJB77P1:527:H7RDTADXX:1:1101:4220:2204 1:N:0:
CGACGCCTGCATAAGGGCTTTCGCTGATTGCCGCGCCCAGGCCCAGGCTGAAATCGCCCGGTTCAGCGTGGGCGAACGGCAACGACAGGGCAGGCAAAGCAACAACGGTCAGTGCGGGAAGGAAAAAGTGTTTCATGACGGCGG
+
DDHHGHGJIJJJJJHIJJJJJJIIIJJJEIIIGIEIJHHEFDFDDDDDDDDDDDDDDDD@B5;<ACC9@BD@?BD<BDDDBDBDBBDBDB<?BBDDBDCDDDBDDDDD@@BD@ACDDDDDDBDDDDDD:::@CC@C@CDBBBBD
@DJB77P1:527:H7RDTADXX:1:1101:4220:2204 3:N:0:
AGCTTATACACGCCAGAAAGATCACTCAGAGAGCCGCCGTCATGAAACACTTTTTCCTTCCCGCAC
+
=DDDFFHHHGGIIIIJJJJJIJIIIIIJFIIIJJJIJIJIFGIJIJJGGIHGHGHGFFFEFFDDDD
@DJB77P1:527:H7RDTADXX:1:1101:4290:2210 1:N:0:
CTGTACTCGATCGGCAAGGATTCGGCCGTGATGCTGCACCTGGCGCGCAAGGCTTTCTTCCCCGGCAAACTGCCATTCCCTGTGATGCATGTCGATACCCGCTGGAAATTCCAGGAGATGTATCGCTTTCGCGACCAGATGGTC
+
FFHFDHHJIBGIGIGIJJJIJJIIJIGIDGBHIIIGEAHIGJGGAHFDDD?BDDDC>CACCDCDDDBBDDDDDDDCCADDDDCDEECDDEC@CAB?ABDABD<BBDCCDCDDDCDBDBCCC>@BBB@D0><9<>BDDD8@ACCC
@DJB77P1:527:H7RDTADXX:1:1101:4290:2210 3:N:0:
TCGACATGCATCACAGGGAATGGCAGTTTGCCGGGGAAGAAAGCCTTGCGCGCCAGGTGCAGCATCACGGCCGAATCCT
+
?BDA@BBCCDEDDDDDDDDDDDDDDDDDEDDDDDDDBBDDDDDDDDDDDDDDDDDDD:A>CCABDDDDDDDDDDDDDCC
@DJB77P1:527:H7RDTADXX:1:1101:6926:2244 1:N:0:
ATATCGTATTTCCAGAGTGCCACTGGCTGATAAAGAAAGAGGCCGGGCATATGCTGATTCTGGCTATTTACCTTGACGCTCGCAGCAGCGATATCGACGGATCAGGGTTTAAATCATCCTCGTATTTAACTTTGGGAAAACAAC
+
FFGHHHHJJJJJJJJJJFHIJJJJJIJJJIJJJIIIJJGIIJIJIJJJIIJJJJIHHHHGHHFFFFFEEEEEEEDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDEDDDDDDDD<BDDEDEDDEDDCDDDDDDDDD
@DJB77P1:527:H7RDTADXX:1:1101:6926:2244 3:N:0:
GGAGCTACCTTCCTGAACCAGGCGCATCACGTGTGTTTTTGACACCGATAGTTGTTTTCCCAAAGTTAAATACGAGGATGATTTAAACCCTGATCCGTCGATATCGCTGCTGCGAGCGTCAAGGTAAATAGCCAGAATCAGCATATGCCC
+
CCCFFFFFGHHHHJJJJJJIGIJJJJIIJJJHHGGHIJJJJJJJIIJGIIFHHHHFFFFFDEEEDEDDEDDECDDDDDDDDEEEDEDDDDDDDDDDDBDDDDBDDDDDDDDDDDDDDDBDDDDD>>CC@DACDDDDDDDDDDDCCDECDD
@DJB77P1:527:H7RDTADXX:1:1101:9747:2206 1:N:0:
TGCTCGTCAGGTACTCGCTGAGGTTGTACAACTGCGCGATGTCGTTGCCATCGGCCATCAACACGCTACTGCGTACGTAGCCGCCGTTGAGCCACTCAAAGCTGTCGTTACCGAAGCTTGCGCGTATTTCTCCACGAACTTCGC
+
FFHHHHHJJIJIHIJJJJJJJJJJJJJJIJJJJJGJJJJJJIJIJHEHHHFFFDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDBDDDDDDBDDDBDDDEEEDDDDDDDDDDD?B
@DJB77P1:527:H7RDTADXX:1:1101:9747:2206 3:N:0:
AACGTCTTCGAGCTTTCTGGCGGCAGCATTATCAACAATCTGGTCGCCGGGTTTGGCCGGGACACGATTACAGTGTCGG
+
8@<@<ACBDDDDBDDDDDCDCDDBDDDDCDCCCDDCCDC?ACC@CBDDDDDBBD?B>CDDD>BDBD3<?<@AC>>@@BB
@DJB77P1:527:H7RDTADXX:1:1101:12008:2199 1:N:0:
CACTGCGGCCGCCTGCGATGAAGTTCTGGCCGAGAATGTTAGGCATCGGTATCTCCTGTTGTTTGATGTTGGAGGCGGGCGCCGAAGGGATCCGGACCGTGCGCTCCTTATGGGTTGCCGAGTTGCTTTGCGGGGTGGCACTGG
+
FFHHHHHJJJJJJJJJJHJJJJJJJJJJJJJJHJIHHHHHHFFFFFEDDDDDDFDDDDDDDDDDDCDDDDDDCDDDDCDBBDDB9B9BDB<<C<B>@@.5?B>@B>@CC>C>CD3<9<A9<B@BCA>ACAC<>BD9BD@A@<C@
@DJB77P1:527:H7RDTADXX:1:1101:12008:2199 3:N:0:
AGCTTAAACAGCAGTGGCGACATAAAGGCTAAACGCTGTCTGGCAGTTGCGTCAGCAGCGAATCAA
+
=DDFFFHHHHHJIIIJJJJJJJJJJJJJGIIJJJJIIJGIGIIIEIIIIJJJHFHFFFFDDDDDDD
@DJB77P1:527:H7RDTADXX:1:1101:14057:2199 1:N:0:
AGCCGGAAATAAGCAACGTCCGATCAATCCTGTCACAGGGTTTGGTGGCAGTGATCGCGCTGGCTCTCGATGGTGAAGGTTCAATGAGGCATTTGAAGATCAAGAGCCTTTCCCTTACCGGACATGACGGACCCGGTATACGCG
+
FFHHHHHJIJJJJJJJJFHIIJIJJJJJJJJJJJJJJJJJHIJJJIHHHHFFFFFEDDDDDDDDDDDDDDDEDACDDDDCCDDEEDDDDDDDDDEEDDDDDDDDDDDDDDDDDDDDDDDDDDBDDDDDDDDDDDDDBDDEEDDD
@DJB77P1:527:H7RDTADXX:1:1101:14057:2199 3:N:0:
AGCTATTGATATTCCAAATCTTGGGTAAGCATCACGATCTGATTGGATCGGATCTTCTCGCGTATA
+
=DDFFFHHHHHJJJJJJJJJJJJJJHHIJJJJJ@GIJIJJJIJJJJIJHIJJJJIJJJIIJJHHFF
@DJB77P1:527:H7RDTADXX:1:1101:14176:2213 1:N:0:
CCTTGGCTTCGGCGTGCGCCGAGAAGGCGGGGATCATCAAGGCCAGAAAAATGGCATGAAATTTCATCGGTGCTCCTGCGGATGAATATGACTTCATTGGATCCAAAATTTACTCCAGTCGCGTGCCTCTGACTAGCGGGTGCC
+
FFHHHHHJJJJJJJIJJJJJJJJJJJJJJHDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDEEEDDBBDDDDDDDDDBDDDDEEEEEDDDDDEEDDDDDDDDDDDDDDEDDDDDDDDDDDDDDDDDDDDDCCDDDDBBBD
@DJB77P1:527:H7RDTADXX:1:1101:14176:2213 3:N:0:
AGCTAGAACAACAGCGATTGCCGGAATCAACCCGCGCCAGGCAAGGTCAGCCAACATGACAACCCGACGAAAGGCCACTGGCACCCGCTAGTCAGAGGCACGCGACTGGAGTAAATTTTGGATCCAATGAAGTCATATTCATCCGCAG
+
=DDFFFHHHHHJJIJIJJJJJJJJJJJJJJJJJJJJJJJJHHHHEF@DFEEEEEDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDCDEDDDDDBDDDDDDDDDDD>CCDDEDDDDDDDDDDDDDDDCDEEEEEEEEEDDDDC
@DJB77P1:527:H7RDTADXX:1:1101:15053:2181 1:N:0:
CAGCTCAGGAGGATTTTCCCGCGTGTCGGCCACAGCAAAGGACACGCCCCGGTTCGCCAGGAAGCGAACCAGGGACATGCCACTCTTGCCAAGCCCCACAACAATGCGGAAGCGGTCGGAAACGATCAGGGACACTCGTTCTAC
+
FFHHHHHJJJJJJJJJJJJJIJJJJJJJJJJJJJJJJJJIJJJHHHFFDDBD?BDDDDDDDDDDDDDDDDDBDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDCDDDDDDDDBDDDBDDDDDDDDDDDDDDDCDDDDDDED
@DJB77P1:527:H7RDTADXX:1:1101:15053:2181 3:N:0:
CAGTGGTCATTCAGGTTGCGTCTTTCAA
+
<@@?@?@?@???????????????????
@DJB77P1:527:H7RDTADXX:1:1101:15413:2202 1:N:0:
GTGCAATTGCCGACCTGCTGGTGGTGGACGGCGACCCGCTGAGCGATATCAGCTGCCTGGTGGGCCAGGGCGAGCAGTTGGCGATGATTGTTCAGGGTGGTCATGTGCACAAGAACACCCTGGCCTGATCAGCCGTCAGTCGGC
+
FDHGHHHIIIJJIJJIJJIJGFHIDHIHIIJJIJJHFEDDD@DDDDDDDDEEDDDDCDDD@CDDDDDDDDBDDDDDDCCDDDBBDDDEEDDDDEDDDCDD>CCDDCDDEDDDDDDDDDDDDDDDDDDDDDEDDDDBBBDDDBBB
@DJB77P1:527:H7RDTADXX:1:1101:15413:2202 3:N:0:
CAGGCCAGGGTGTTCTTGTGCACATGACCACCCTGAACAATCATCGCCAACTGCTCGCCCTGGCCCACCAGGCAGCTGA
+
?BDDDDDDDD<?@DDEDDDDDEDDDDDDDDBDDDCDDDCCDCDDDDBDDDBDCDDDBBBDBBDDDDDDDDBBDDDDDCC
@DJB77P1:527:H7RDTADXX:1:1101:15955:2211 1:N:0:
GTGGCTTCCGTTCCGCCTCAAGGTGGTCGCAAGCATCCGCAGCAGGAATTCCTGCAGGTGGACACGCGCAACATCCTCTTCATATGCGGTGGCGCCTTCTCGGGCTTGGAAAAGGTCATTCAGAACCGTTCCACCCGTGGCGGC
+
FFHHGHHIGIIIJHIIJIJJJIICGGHIIIJIIIIJJJJJJIGIFHGHFFEFFEEEEEEDDDDDDDDDDDDDBDCDDDDDDEEEEEDDDBDBDDB>BDDDDDDDDDDDDDDCCDD@CDDEEECDDDDDBBDDDDDDD<@BDBDD
@DJB77P1:527:H7RDTADXX:1:1101:15955:2211 3:N:0:
AGCTCTCCTTGCTGCGAACTTCGGCGTTGAAACCGATGCCGCCACGGGTGGAACGGTTCTGAATGACCTTTTCCAAGCCCGAGAAGGCGCCACCGCATATGAAGAGGATGTTGCGCGTGTCCACCTGCAGGAATTCCTGCTGCGGATG
+
=DDFFEHFHHHJJJJJGHIIJIIIJJJJJIIJIJJIGIJJJHFFFDDD9?B?CCBD?BBCDDDDDDDDDDDDDCEDDDDDDDDDDDDDDDDDDDDDDDDACDEDABDBDDDDDCBDDDDDDDDDDDDDDDDBDDDDDDDDDDDDDDDD
@DJB77P1:527:H7RDTADXX:1:1101:16246:2219 1:N:0:
CTATATAGACCTGCGCTCTTGGCCAAGTCGATGCCTTGCCGCTGTCGCTCTCGCCGGTCCTCGAAGTCGTCACGCGCTATTTGCAGTGCCACACGCAACAGCATGTCTTGTACGCCTTGCAACACCACCTTGGCGACGCCACTG
+
FFHHHHHJJJJJJJJJJJJJIIIJJJJJJJJJJJJJJJJJJJJJJJJJJJJHHHFDDDDDDDDDDDDDDDDDDDDDDDDDDEDDEDDDDDDDDDDDDDDDDDDDDDDEDDDDEDDDDDDDDDDDDDDDDDDCDCDDDDDDDDCC
@DJB77P1:527:H7RDTADXX:1:1101:16246:2219 3:N:0:
GGAGCTGTGAAGTCGTCATCGCTGAGAAGATCGACCGAATCAGCCGTCTTCCCTTGGTCGAAGCAGAAAGGCTTGTGGACGCGATCAAGGCTAAGGGCGCACGCTTGGCGGTGCCAGGCATCGTCGATTTATCGGAATTGGCCGAGGCAT
+
CC@FFFFFHHHHHIJJJJJIJJJJJJJJJJJJJJJJJJJJIJJJJJJJJHGHHHFFFFFDDDDDDDDDDDDDDDDBDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDBDDDBDDDDDDDDDCDBDD>BDDCDCBD?B?CC@CDD<BBDBC
@DJB77P1:527:H7RDTADXX:1:1101:18918:2216 1:N:0:
GGGCCGTCCGGGGCAGGAAAAGAAGTCATCGCCCAGCGCTTGCATCGGCTTGGGGTGAACCCGCTCCATCCCTTTGTGGATATCAACTGCGCTGCATTGCCAGCACACCTCATTGAAGCAGAGCTATTCGGTCATAGCCGAGGT
+
FFHHHHDHHHIJJJJJJIIJJJJJJGIJJJJJJJHHHFFCDDCCDDDDDDDDDDD)5?C@BD5955;::>AB:ACC@?B:CDDCDCC:>C<><<>>CDACC>@?9???BCA:>@:4@>AC9?8AACCC>9A8<5>B@CC959@9

[Bioinform]

Thanks Brian. Have a gr8 weekend.

[Brian Bushnell]

OK, I looked at the code - your fastq file is corrupt; you are missing a "+" symbol on a line. It's probably truncated. You should go back to the raw reads and reprocess them.

@Brian: @Bioinform appears to be submitting under slurm so I am surprised that it is allowing BBMap to grab all cores (perhaps @Bioinform is specifying them on other part of the command line).

Oh, that's good to know; I've never used Slurm.

[Bioinform]

@ GenoMax

Thanks, will match it.

@Brian

with bbduk.sh also i am getting error messages.

Module slurm/15.08.1 loaded
java -Djava.library.path=/home/software/BBMap/bbmap/jni/ -ea -Xmx6519m -Xms6519m -cp /home/software/BBMap/bbmap/current/ jgi.BBDuk2 in=GGAGC_trimmed2.fastq trimq=6 out=407_PST.fastq
Executing jgi.BBDuk2 [in=GGAGC_trimmed2.fastq, trimq=6, out=407_PST.fastq]

BBDuk2 version 35.66

NOTE: No reference files specified, no trimming mode, no min avg quality, no histograms - read sequences will not be changed.
Initial:
Memory: max=6550m, free=6482m, used=68m

Input is being processed as unpaired
Started output streams: 0.054 seconds.
java.lang.ArrayIndexOutOfBoundsException: 0
at stream.FASTQ.quadToRead(FASTQ.java:723)
at stream.FASTQ.toReadList(FASTQ.java:653)
at stream.FastqReadInputStream.fillBuffer(FastqReadInputStream.java:111)
at stream.FastqReadInputStream.nextList(FastqReadInputStream.java:96)
at stream.ConcurrentGenericReadInputStream$ReadThread.readLists(ConcurrentGenericReadInputStream.java:659)
at stream.ConcurrentGenericReadInputStream$ReadThread.run(ConcurrentGenericReadInputStream.java:638)
Exception in thread "Thread-16" Exception in thread "Thread-15" java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
Exception in thread "Thread-19" java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
Exception in thread "Thread-4" Exception in thread "Thread-11" java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
Exception in thread "Thread-8" java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
Exception in thread "Thread-5" Exception in thread "Thread-17" Exception in thread "Thread-7" java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
Exception in thread "Thread-6" java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
Exception in thread "Thread-12" Exception in thread "Thread-9" Exception in thread "Thread-10" Exception in thread "Thread-13" java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
Exception in thread "Thread-18" java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
java.lang.NullPointerException
at jgi.BBDuk2$ProcessThread.run(BBDuk2.java:2417)
Processing time: 0.110 seconds.

Input: 5200 reads 724560 bases.
Result: 5175 reads (99.52%) 724439 bases (99.98%)

Time: 0.176 seconds.
Reads Processed: 5200 29.58k reads/sec
Bases Processed: 724k 4.12m bases/sec
Exception in thread "main" java.lang.RuntimeException: jgi.BBDuk2 terminated in an error state; the output may be corrupt.
at jgi.BBDuk2.process(BBDuk2.java:888)
at jgi.BBDuk2.main(BBDuk2.java:70)

Thanks

[GenoMax]

@Brian: @Bioinform appears to be submitting under slurm so I am surprised that it is allowing BBMap to grab all cores (perhaps @Bioinform is specifying them on other part of the command line).

The error appears to be happening in first few reads if I am reading the stats right.

EDIT: @Bioinform: Add threads=8 to match your request of 8 cores (-n 8) via slurm (that won't fix the problem but just to be consistent).

[Bioinform]

I used as batch with following

#!/bin/bash -l
#SBATCH -J bbsplit
#SBATCH -o myjob.%A_%a.output
#SBATCH -e myjob.%A_%a.error
#SBATCH -p serial
#SBATCH -t 999:00:00
#SBATCH -N 1
#SBATCH -n 8
#SBATCH --mem 20000

echo start

bbsplit.sh in=/home/Whole_genome_analysis/HiseQ/HiseQ_data/PST/Trimmed_Seq/GGAGC_trimmed1.fastq ref=/home/spthapa/Whole_genome_analysis/Reference/NCPPB382/Cmm382_genome.fasta,/home/Whole_genome_analysis/Reference/NCPPB382/Xcitri_jx6.fasta basename=PST_407_%.fq outu=PST_407_unmapped.fq

echo finished

I also ran directly using
bbsplit.sh in=/home/Whole_genome_analysis/HiseQ/HiseQ_data/PST/Trimmed_Seq/GGAGC_trimmed1.fastq ref=/home/spthapa/Whole_genome_analysis/Reference/NCPPB382/Cmm382_genome.fasta,/home/Whole_genome_analysis/Reference/NCPPB382/Xcitri_jx6.fasta basename=PST_407_%.fq outu=PST_407_unmapped.fq

I get the same error.

I am not sure of that.

Thanks

[Brian Bushnell]

BBMap autodetects the number of processors available and uses all of them, by default. This looks to me like a corrupt fastq file (GGAGC_trimmed1.fastq). I'll have to look at the specifics... but probably, either some reads were trimmed to zero length and I am not correctly handling that (so it is a bug), or the file is corrupt and some other lines (like the header or the line with the +) are zero length.

I'll get back to you some time later today. If it's a bug due to zero-length reads, I'll post a fixed version on Monday, but in the mean time you can circumvent that by discarding zero-length reads when trimming, or trimming to a minimum length of 1 (which is what BBDuk does, to avoid zero-length reads).