Unrecognized command when running unicycler_polish

[mradz19]

I am trying to run unicycler_polish on my assembled seqeuences, however the program is failing during the "Pilon polish, large variants, ALE assessed" stage, with the following error:

Round 3: Pilon polish, large variants, ALE assessed
2019-05-20 14:30:30 bowtie2-build 001_2_polish.fasta bowtie_index
2019-05-20 14:30:30 bowtie2 --local --very-sensitive-local --threads 6 -I 101 -X 708 -x bowtie_index -1 ../../../../Short_reads/Enterococcus_reads/faecalis/MB19052739.2_paired_R1.fastq -2 ../../../../Short_reads/Enterococcus_reads/faecalis/MB19052739.2_paired_R2.fastq | /anaconda3/bin/samtools view -hu - | /anaconda3/bin/samtools sort -@ 6 -o illumina_alignments.bam -
2019-05-20 14:31:04 samtools index illumina_alignments.bam
2019-05-20 14:31:05 java -jar /Users/michaelradzieta/nanopore_software/pilon-1.23.jar --genome 001_2_polish.fasta --frags illumina_alignments.bam --fix local --changes --outdir temp_pilon
2019-05-20 14:31:16 cp temp_pilon/pilon.changes 003_1_pilon.changes
2019-05-20 14:31:16 rm bowtie_index.1.bt2 bowtie_index.2.bt2 bowtie_index.3.bt2 bowtie_index.4.bt2 bowtie_index.rev.1.bt2 bowtie_index.rev.2.bt2 illumina_alignments.bam illumina_alignments.bam.bai
2019-05-20 14:31:16 rm -r temp_pilon

2019-05-20 14:31:16 bowtie2-build 001_2_polish.fasta bowtie_index
2019-05-20 14:31:16 bowtie2 --end-to-end --very-sensitive --threads 6 -I 101 -X 708 -x bowtie_index -1 ../../../../Short_reads/Enterococcus_reads/faecalis/MB19052739.2_paired_R1.fastq -2 ../../../../Short_reads/Enterococcus_reads/faecalis/MB19052739.2_paired_R2.fastq | /anaconda3/bin/samtools view -hu - | /anaconda3/bin/samtools sort -@ 6 -o illumina_alignments.bam -
2019-05-20 14:31:34 samtools index illumina_alignments.bam
2019-05-20 14:31:35 samtools --nout --metagenome illumina_alignments.bam 001_2_polish.fasta ale.out
[main] unrecognized command '--nout'

This results in the termination of unicycler_polish. Has anyone had this issue?

[dswan]

The arguments being passed to samtools "--nout --metagenome illumina_alignments.bam 001_2_polish.fasta ale.out" are meant to be passed to ALE.

Are you sure sure you specified the right location for ALE with --ale? It looks like you might have inadvertently passed it the location of samtools

[mradz19]

@dswan that may be the problem. I was having trouble installing ALE, to my understanding it is included as part of samtools? That's why I directed it to the samtools executable. Is that incorrect?

[dswan]

I don't think ALE has ever been a part of samtools, and is available here: https://github.com/sc932/ALE

It compiles and runs just fine on my fairly standard Ubuntu environment.

[rrwick]

No longer relevant for the current version (see #226), so closing now.