snippy: Error running command

[monicanielsen]

I am trying to run snippy, but i get the following:

(base) sfb619@SUND35882 snippy % snippy --cpus 4 --outdir mysnips --ref 26B3_contigs.fsa --R1 AL8_S42_L001_R1_001.fastq.gz --R2 AL8_S42_L001_R2_001.fastq.gz --ram 3

[21:37:19] This is snippy 4.6.0 [21:37:19] Written by Torsten Seemann [21:37:19] Obtained from https://github.com/tseemann/snippy [21:37:19] Detected operating system: darwin [21:37:19] Enabling bundled darwin tools. [21:37:19] Found bwa - /usr/local/bin/bwa [21:37:19] Found bcftools - /usr/local/bin/bcftools [21:37:19] Found samtools - /usr/local/bin/samtools [21:37:19] Found java - /usr/bin/java [21:37:19] Found snpEff - /usr/local/bin/snpEff [21:37:19] Found samclip - /usr/local/bin/samclip [21:37:19] Found seqtk - /usr/local/bin/seqtk [21:37:19] Found parallel - /usr/local/bin/parallel [21:37:19] Found freebayes - /usr/local/bin/freebayes [21:37:19] Found freebayes-parallel - /usr/local/bin/freebayes-parallel [21:37:19] Found fasta_generate_regions.py - /usr/local/bin/fasta_generate_regions.py [21:37:19] Found vcfstreamsort - /usr/local/bin/vcfstreamsort [21:37:19] Found vcfuniq - /usr/local/bin/vcfuniq [21:37:19] Found vcffirstheader - /usr/local/bin/vcffirstheader [21:37:19] Found gzip - /usr/bin/gzip [21:37:19] Found vt - /usr/local/bin/vt [21:37:19] Found snippy-vcf_to_tab - /usr/local/Cellar/snippy/4.6.0/bin/snippy-vcf_to_tab [21:37:19] Found snippy-vcf_report - /usr/local/Cellar/snippy/4.6.0/bin/snippy-vcf_report [21:37:19] Checking version: samtools --version is >= 1.7 - ok, have 1.13 [21:37:19] Checking version: bcftools --version is >= 1.7 - ok, have 1.13 [21:37:19] Checking version: freebayes --version is >= 1.1 - ok, have 1.3.2 [21:37:19] Checking version: snpEff -version is >= 4.3 - ok, have 4.3 [21:37:19] Checking version: bwa is >= 0.7.12 - ok, have 0.7.17 [21:37:19] Using reference: /Users/sfb619/Documents/speciale/snippy/26B3_contigs.fsa [21:37:19] Treating reference as 'fasta' format. [21:37:19] Will use 4 CPU cores. [21:37:19] Using read file: /Users/sfb619/Documents/speciale/snippy/AL8_S42_L001_R1_001.fastq.gz [21:37:19] Using read file: /Users/sfb619/Documents/speciale/snippy/AL8_S42_L001_R2_001.fastq.gz [21:37:19] Creating folder: mysnips [21:37:19] Changing working directory: mysnips [21:37:19] Creating reference folder: reference [21:37:19] Extracting FASTA and GFF from reference. [21:37:20] Wrote 250 sequences to ref.fa [21:37:20] Wrote 0 features to ref.gff [21:37:20] Freebayes will process 7 chunks of 694809 bp, 4 chunks at a time. [21:37:20] Using BAM RG (Read Group) ID: mysnips [21:37:20] Running: samtools faidx reference/ref.fa 2>> snps.log [21:37:20] Running: bwa index reference/ref.fa 2>> snps.log [21:37:21] Running: mkdir -p reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa 2>> snps.log [21:37:21] Running: ln -sf reference/ref.fa . 2>> snps.log [21:37:21] Running: ln -sf reference/ref.fa.fai . 2>> snps.log [21:37:21] Running: mkdir -p reference/ref && gzip -c reference/ref.gff > reference/ref/genes.gff.gz 2>> snps.log [21:37:21] Running: bwa mem -Y -M -R '@RG\tID:mysnips\tSM:mysnips' -t 4 reference/ref.fa /Users/sfb619/Documents/speciale/snippy/AL8_S42_L001_R1_001.fastq.gz /Users/sfb619/Documents/speciale/snippy/AL8_S42_L001_R2_001.fastq.gz | samclip --max 10 --ref reference/ref.fa.fai | samtools sort -n -l 0 -T /private/var/folders/zh/yt6c034s4rv2hl36n49vrtl8h8xs39/T --threads 1 -m 1500M | samtools fixmate -m --threads 1 - - | samtools sort -l 0 -T /private/var/folders/zh/yt6c034s4rv2hl36n49vrtl8h8xs39/T --threads 1 -m 1500M | samtools markdup -T /private/var/folders/zh/yt6c034s4rv2hl36n49vrtl8h8xs39/T --threads 1 -r -s - - > snps.bam 2>> snps.log [M::bwa_idx_load_from_disk] read 0 ALT contigs [samclip] samclip 0.4.0 by Torsten Seemann (@torstenseemann) [samclip] Loading: reference/ref.fa.fai [samclip] Found 250 sequences in reference/ref.fa.fai [M::process] read 167002 sequences (40000285 bp)... [M::process] read 167206 sequences (40000239 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (14, 79612, 11, 5) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (266, 371, 599) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1265) [M::mem_pestat] mean and std.dev: (400.92, 207.32) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1598) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (323, 432, 560) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1034) [M::mem_pestat] mean and std.dev: (446.19, 185.14) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1271) [M::mem_pestat] analyzing insert size distribution for orientation RF... [M::mem_pestat] (25, 50, 75) percentile: (4649, 4817, 4909) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (4129, 5429) [M::mem_pestat] mean and std.dev: (4856.62, 113.46) [M::mem_pestat] low and high boundaries for proper pairs: (3869, 5689) [M::mem_pestat] skip orientation RR as there are not enough pairs [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RF [M::mem_process_seqs] Processed 167002 reads in 13.332 CPU sec, 3.205 real sec [samclip] Processed 100000 records... [M::process] read 166864 sequences (40000130 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (14, 79668, 9, 9) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (253, 358, 764) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1786) [M::mem_pestat] mean and std.dev: (406.92, 267.45) [M::mem_pestat] low and high boundaries for proper pairs: (1, 2297) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (323, 431, 558) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1028) [M::mem_pestat] mean and std.dev: (444.49, 184.37) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1263) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] skip orientation RR as there are not enough pairs [M::mem_pestat] skip orientation FF [M::mem_process_seqs] Processed 167206 reads in 13.989 CPU sec, 3.287 real sec [samclip] Processed 200000 records... [samclip] Processed 300000 records... [M::process] read 166698 sequences (40000161 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (19, 79449, 11, 6) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (154, 354, 526) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1270) [M::mem_pestat] mean and std.dev: (325.65, 194.18) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1642) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (326, 433, 562) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1034) [M::mem_pestat] mean and std.dev: (447.25, 183.73) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1270) [M::mem_pestat] analyzing insert size distribution for orientation RF... [M::mem_pestat] (25, 50, 75) percentile: (4659, 4821, 4892) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (4193, 5358) [M::mem_pestat] mean and std.dev: (4792.36, 150.14) [M::mem_pestat] low and high boundaries for proper pairs: (3960, 5591) [M::mem_pestat] skip orientation RR as there are not enough pairs [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RF [M::mem_process_seqs] Processed 166864 reads in 13.824 CPU sec, 3.255 real sec [samclip] Processed 400000 records... [samclip] Processed 500000 records... [M::process] read 166496 sequences (40000444 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (12, 79429, 6, 4) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (221, 272, 305) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (53, 473) [M::mem_pestat] mean and std.dev: (235.73, 90.25) [M::mem_pestat] low and high boundaries for proper pairs: (1, 597) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (329, 438, 575) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1067) [M::mem_pestat] mean and std.dev: (456.66, 193.17) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1313) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] skip orientation RR as there are not enough pairs [M::mem_pestat] skip orientation FF [M::mem_process_seqs] Processed 166698 reads in 14.708 CPU sec, 3.446 real sec [samclip] Processed 600000 records... [M::process] read 87066 sequences (20932223 bp)... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (14, 79228, 14, 2) [M::mem_pestat] analyzing insert size distribution for orientation FF... [M::mem_pestat] (25, 50, 75) percentile: (128, 345, 1781) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5087) [M::mem_pestat] mean and std.dev: (684.14, 722.11) [M::mem_pestat] low and high boundaries for proper pairs: (1, 6740) [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (329, 439, 574) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1064) [M::mem_pestat] mean and std.dev: (456.29, 192.22) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1309) [M::mem_pestat] analyzing insert size distribution for orientation RF... [M::mem_pestat] (25, 50, 75) percentile: (4442, 4747, 4859) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (3608, 5693) [M::mem_pestat] mean and std.dev: (4780.64, 170.40) [M::mem_pestat] low and high boundaries for proper pairs: (3191, 6110) [M::mem_pestat] skip orientation RR as there are not enough pairs [M::mem_pestat] skip orientation FF [M::mem_pestat] skip orientation RF [M::mem_process_seqs] Processed 166496 reads in 14.538 CPU sec, 3.485 real sec [samclip] Processed 700000 records... [samclip] Processed 800000 records... [M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (8, 41451, 8, 6) [M::mem_pestat] skip orientation FF as there are not enough pairs [M::mem_pestat] analyzing insert size distribution for orientation FR... [M::mem_pestat] (25, 50, 75) percentile: (327, 436, 573) [M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 1065) [M::mem_pestat] mean and std.dev: (455.42, 191.56) [M::mem_pestat] low and high boundaries for proper pairs: (1, 1311) [M::mem_pestat] skip orientation RF as there are not enough pairs [M::mem_pestat] skip orientation RR as there are not enough pairs [M::mem_process_seqs] Processed 87066 reads in 8.035 CPU sec, 1.936 real sec [samclip] Processed 900000 records... [main] Version: 0.7.17-r1188 [main] CMD: bwa mem -Y -M -R @RG\tID:mysnips\tSM:mysnips -t 4 reference/ref.fa /Users/sfb619/Documents/speciale/snippy/AL8_S42_L001_R1_001.fastq.gz /Users/sfb619/Documents/speciale/snippy/AL8_S42_L001_R2_001.fastq.gz [main] Real time: 19.594 sec; CPU: 79.090 sec [samclip] Total SAM records 924966, removed 109993, allowed 48238, passed 814973 [samclip] Header contained 252 lines [samclip] Done. [21:38:19] Running: samtools index snps.bam 2>> snps.log [21:38:20] Running: fasta_generate_regions.py reference/ref.fa.fai 694809 > reference/ref.txt 2>> snps.log [21:38:20] Running: freebayes-parallel reference/ref.txt 4 -p 2 -P 0 -C 2 -F 0.05 --min-coverage 10 --min-repeat-entropy 1.0 -q 13 -m 60 --strict-vcf -f reference/ref.fa snps.bam > snps.raw.vcf 2>> snps.log [21:38:22] Error running command, check mysnips/snps.log (base) sfb619@SUND35882 snippy %

any suggestions what i can do to make it work? thanks