Comments (5)
Hello Lauren,
Thanks for pointing to the bedtools Version: v2.27.1, after updating from Version: v2.25.0 to this newer version (v2.27.1) now I have the .fa and .bed output after running tigmint-cut.
Thanks for your help !!
from arcs.
Hi @Dharmendra-G-1 -
Could you please enclose copy-and-paste blocks in triple back ticks? Using that formatting will make your pasted commands much easier to read.
(https://help.github.com/articles/basic-writing-and-formatting-syntax/#quoting-code)
Also, can you include the standard output from your the tigmint
command? It looks like something went wrong in the tigmint-cut
command.
from arcs.
Hello Lauren,
Thanks for your response, Sorry for confusion with format: As per your request here are details:
I am using
draft.fasta and rename linked reads as reads.fa.gz
The main error I am getting is from tigmint-cut:
ubuntu@ip-10-68-102-172:/mid_large_2t/running_arcs$ python3 tigmint/bin/tigmint-cut -p 40 -w 1000 -n 20 -t 0 draft.fa draft.reads.as0.65.nm5.molecule.size2000.bed --fastaout draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa
Started at: 2019-01-06 18:04:53.506702
Reading contig lengths...
Finding breakpoints...
Cutting assembly at breakpoints...
Tool: bedtools getfasta (aka fastaFromBed)
Version: v2.25.0
Summary: Extract DNA sequences into a fasta file based on feature coordinates.
Usage: bedtools getfasta [OPTIONS] -fi <fasta> -bed <bed/gff/vcf> -fo <fasta>
Options:
-fi Input FASTA file
-bed BED/GFF/VCF file of ranges to extract from -fi
-fo Output file (can be FASTA or TAB-delimited)
-name Use the name field for the FASTA header
-split given BED12 fmt., extract and concatenate the sequencesfrom the BED "blocks" (e.g., exons)
-tab Write output in TAB delimited format.
- Default is FASTA format.
-s Force strandedness. If the feature occupies the antisense,
strand, the sequence will be reverse complemented.
- By default, strand information is ignored.
-fullHeader Use full fasta header.
- By default, only the word before the first space or tab is used.
DONE!
Ended at: 2019-01-06 18:16:46.167144
The initial command I ran are as follows:
./arcs-master/Examples/arcs-make arcs-tigmint draft=draft reads=reads m=40
which ran this
./arcs-master/Examples/arcs-make arcs-tigmint -n draft=draft reads=reads m=40
ln -s draft.fasta draft.fa
./tigmint/bin/tigmint tigmint draft=draft reads=reads minsize=2000 as=0.65 nm=5 dist=50000 mapq=0 trim=0 span=20 window=1000 t=40
touch empty.fof
perl -ne 'chomp; if(/>/){$ct+=1; print ">$ct\n";}else{print "$_\n";} ' < draft.tigmint.fa > draft.tigmint.renamed.fa
bwa index draft.tigmint.renamed.fa
sh -c 'bwa mem -t40 -C -p draft.tigmint.renamed.fa reads.fq.gz | /mid_large_2t/important/samtools-1.6/samtools view -Sb - | /mid_large_2t/important/samtools-1.6/samtools sort -@40 -n - -o draft.tigmint.sorted.bam'
echo draft.tigmint.sorted.bam > draft.tigmint_bamfiles.fof
./arcs-master/Arcs/arcs --bx -v -f draft.tigmint.renamed.fa -a draft.tigmint_bamfiles.fof -c 5 -m 40 -s 98 -r 0.05 -e 30000 -z 500 -d 0 --gap 100 -b draft.tigmint_c5_m40_s98_r0.05_e30000_z500
python /mid_large_2t/running_arcs/arcs-master/Examples/makeTSVfile.py draft.tigmint_c5_m40_s98_r0.05_e30000_z500_original.gv draft.tigmint_c5_m40_s98_r0.05_e30000_z500.tigpair_checkpoint.tsv draft.tigmint.renamed.fa
ln -s draft.tigmint_c5_m40_s98_r0.05_e30000_z500.tigpair_checkpoint.tsv draft.tigmint_c5_m40_s98_r0.05_e30000_z500_l5_a0.3.tigpair_checkpoint.tsv
/opt/links_v1.8.6/LINKS -f draft.tigmint.renamed.fa -s empty.fof -b draft.tigmint_c5_m40_s98_r0.05_e30000_z500_l5_a0.3 -l 5 -a 0.3 -z 500
rm draft.tigmint_c5_m40_s98_r0.05_e30000_z500_l5_a0.3.tigpair_checkpoint.tsv
I believe the Tigmint tigmint-make will run as follows from the starting part of arcs-tigment
./tigmint tigmint draft=draft reads=reads minsize=2000 as=0.65 nm=5 dist=50000 mapq=0 trim=0 span=20 window=1000 t=40
./tigmint/bin/tigmint tigmint -n draft=draft reads=reads minsize=2000 as=0.65 nm=5 dist=50000 mapq=0 trim=0 span=20 window=1000 t=40
bwa index draft.fa
bwa mem -t40 -pC draft.fa reads.fq.gz | /mid_large_2t/important/samtools-1.6/samtools view -u -F4 | /mid_large_2t/important/samtools-1.6/samtools sort -@40 -tBX -m 2G -T tmp/ -o draft.reads.sortbx.bam
tigmint/bin/tigmint-molecule -a0.65 -n5 -q0 -d50000 -s2000 draft.reads.sortbx.bam | sort -k1,1 -k2,2n -k3,3n >draft.reads.as0.65.nm5.molecule.size2000.bed
/mid_large_2t/important/samtools-1.6/samtools faidx draft.fa
tigmint/bin/tigmint-cut -p40 -w1000 -n20 -t0 -o draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa draft.fa draft.reads.as0.65.nm5.molecule.size2000.bed
ln -sf draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa draft.tigmint.fa
The tail end of error message I got is as follows:
[M::mem_pestat] mean and std.dev: (242.76, 127.77)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 849)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_process_seqs] Processed 2619274 reads in 1927.504 CPU sec, 50.692 real sec
[main] Version: 0.7.15-r1140
[main] CMD: bwa mem -t40 -pC draft.fa reads.fq.gz
[main] Real time: 52106.558 sec; CPU: 1897517.768 sec
[bam_sort_core] merging from 440 files and 40 in-memory blocks...
tigmint/bin/tigmint-molecule -a0.65 -n5 -q0 -d50000 -s2000 draft.reads.sortbx.bam | sort -k1,1 -k2,2n -k3,3n >draft.reads.as0.65.nm5.molecule.size2000.bed
/mid_large_2t/important/samtools-1.6/samtools faidx draft.fa
tigmint/bin/tigmint-cut -p40 -w1000 -n20 -t0 -o draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa draft.fa draft.reads.as0.65.nm5.molecule.size2000.bed
Started at: 2019-01-05 19:06:03.814077
Reading contig lengths...
Finding breakpoints...
Tool: bedtools getfasta (aka fastaFromBed)
Version: v2.25.0
Summary: Extract DNA sequences into a fasta file based on feature coordinates.
Usage: bedtools getfasta [OPTIONS] -fi <fasta> -bed <bed/gff/vcf> -fo <fasta>
Options:
-fi Input FASTA file
-bed BED/GFF/VCF file of ranges to extract from -fi
-fo Output file (can be FASTA or TAB-delimited)
-name Use the name field for the FASTA header
-split given BED12 fmt., extract and concatenate the sequencesfrom the BED "blocks" (e.g., exons)
-tab Write output in TAB delimited format.
- Default is FASTA format.
-s Force strandedness. If the feature occupies the antisense,
strand, the sequence will be reverse complemented.
- By default, strand information is ignored.
-fullHeader Use full fasta header.
- By default, only the word before the first space or tab is used.
Cutting assembly at breakpoints...
DONE!
Ended at: 2019-01-05 19:17:49.508013
ln -sf draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa draft.tigmint.fa
make[1]: Leaving directory '/mid_large_2t/running_arcs'
touch empty.fof
perl -ne 'chomp; if(/>/){$ct+=1; print ">$ct\n";}else{print "$_\n";} ' < draft.tigmint.fa > draft.tigmint.renamed.fa
bwa index draft.tigmint.renamed.fa
[bwa_index] Pack FASTA... [bns_fasta2bntseq] Failed to allocate 0 bytes at bntseq.c line 303: Success
arcs-master/Examples/arcs-make:157: recipe for target 'draft.tigmint.renamed.fa.bwt' failed
make: *** [draft.tigmint.renamed.fa.bwt] Error 1
the output file I am getting are as follows:
wxrwxrwx 1 ubuntu ubuntu 11 Jan 4 19:03 draft.fa -> draft.fasta
-rw-rw-r-- 1 ubuntu ubuntu 406757 Jan 4 19:40 draft.fa.amb
-rw-rw-r-- 1 ubuntu ubuntu 1411478 Jan 4 19:40 draft.fa.ann
-rw-rw-r-- 1 ubuntu ubuntu 2494337664 Jan 4 19:40 draft.fa.bwt
-rw-rw-r-- 1 ubuntu ubuntu 440254 Jan 5 19:06 draft.fa.fai
-rw-rw-r-- 1 ubuntu ubuntu 623584396 Jan 4 19:40 draft.fa.pac
-rw-rw-r-- 1 ubuntu ubuntu 1247168840 Jan 4 19:51 draft.fa.sa
-rw-rw-r-- 1 ubuntu ubuntu 2526643067 Jan 3 04:49 draft.fasta
-rw-rw-r-- 1 ubuntu ubuntu 697296771 Jan 7 03:20 draft.reads.as0.65.nm5.molecule.size2000.bed
-rw-rw-r-- 1 ubuntu ubuntu 0 Jan 7 04:00 draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa
-rw-rw-r-- 1 ubuntu ubuntu 342869 Jan 7 04:00 draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa.bed
-rw-rw-r-- 1 ubuntu ubuntu 261106244339 Jan 5 12:02 draft.reads.sortbx.bam
lrwxrwxrwx 1 ubuntu ubuntu 77 Jan 5 19:17 draft.tigmint.fa -> draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa
-rw-rw-r-- 1 ubuntu ubuntu 0 Jan 5 19:17 draft.tigmint.renamed.fa
-rw-rw-r-- 1 ubuntu ubuntu 0 Jan 5 19:17 draft.tigmint.renamed.fa.pac
-rw-rw-r-- 1 ubuntu ubuntu 0 Jan 5 19:17 empty.fof
So after the tigmint-molecule I get the bed file " draft.reads.as0.65.nm5.molecule.size2000.bed" which has 16813449 lines but when it ran the tigmint-cut we get .bed file but no .fa file. draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa file get created but completely empty, so no problem with permission as such. The bed file output are as follows :
head draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa.bed
0 0 15517657 0-1
0 15517657 15517670 0-2
0 15517670 17147778 0-3
0 17147778 17147785 0-4
0 17147785 23376074 0-5
0 23376074 23376100 0-6
0 23376100 29696924 0-7
0 29696924 29697326 0-8
0 29697326 29697665 0-9
0 29697665 29697943 0-10
tail draft.reads.as0.65.nm5.molecule.size2000.trim0.window1000.span20.breaktigs.fa.bed
204375 0 33106 204375
204415 0 57665 204415
204459 0 25333 204459
204499 0 64113 204499
204541 0 58322 204541
204581 0 3400 204581
204621 0 53185 204621
204661 0 20990 204661
204701 0 60883 204701
204741 0 113995 204741
Thanks
from arcs.
Thanks @Dharmendra-G-1 -
Please try using a newer version of bedtools
. The issue is that the more recent versions (I am using 2.27.1
) output the fasta to standard out by default, whereas the older version you are using requires an output fasta file specified by -fo
. You can see that the help page is being printed in standard out, indicating that the bedtools getfasta
command isn't running properly as a result.
Take a look at the different help pages:
Tool: bedtools getfasta (aka fastaFromBed)
Version: v2.25.0
Summary: Extract DNA sequences into a fasta file based on feature coordinates.
Usage: bedtools getfasta [OPTIONS] -fi <fasta> -bed <bed/gff/vcf> -fo <fasta>
Options:
-fi Input FASTA file
-bed BED/GFF/VCF file of ranges to extract from -fi
-fo Output file (can be FASTA or TAB-delimited)
-name Use the name field for the FASTA header
-split given BED12 fmt., extract and concatenate the sequencesfrom the BED "blocks" (e.g., exons)
-tab Write output in TAB delimited format.
- Default is FASTA format.
-s Force strandedness. If the feature occupies the antisense,
strand, the sequence will be reverse complemented.
- By default, strand information is ignored.
-fullHeader Use full fasta header.
- By default, only the word before the first space or tab is used.
Tool: bedtools getfasta (aka fastaFromBed)
Version: v2.27.1
Summary: Extract DNA sequences from a fasta file based on feature coordinates.
Usage: bedtools getfasta [OPTIONS] -fi <fasta> -bed <bed/gff/vcf>
Options:
-fi Input FASTA file
-fo Output file (opt., default is STDOUT
-bed BED/GFF/VCF file of ranges to extract from -fi
-name Use the name field for the FASTA header
-name+ Use the name field and coordinates for the FASTA header
-split given BED12 fmt., extract and concatenate the sequences
from the BED "blocks" (e.g., exons)
-tab Write output in TAB delimited format.
- Default is FASTA format.
-s Force strandedness. If the feature occupies the antisense,
strand, the sequence will be reverse complemented.
- By default, strand information is ignored.
-fullHeader Use full fasta header.
- By default, only the word before the first space or tab
is used.
Hope that helps!
Lauren
from arcs.
Excellent - I'm glad you got it working!
from arcs.
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from arcs.