Installing Nullarbor in python environment

[Ebenn]

Hi Matt,
Thx for your response. Here’s the output from ‘prokka’:
ubuntu@ebenn:~/efn$ prokka
Name:
Prokka 1.12 by Torsten Seemann torsten.seemann@gmail.com
Synopsis:
rapid bacterial genome annotation
Usage:
prokka [options] <contigs.fasta>
General:
–help This help
–version Print version and exit
–docs Show full manual/documentation
–citation Print citation for referencing Prokka
–quiet No screen output (default OFF)
–debug Debug mode: keep all temporary files (default OFF)
Setup:
–listdb List all configured databases
–setupdb Index all installed databases
–cleandb Remove all database indices
–depends List all software dependencies
Outputs:
–outdir [X] Output folder [auto] (default ‘’)
–force Force overwriting existing output folder (default OFF)
–prefix [X] Filename output prefix [auto] (default ‘’)
–addgenes Add ‘gene’ features for each ‘CDS’ feature (default OFF)
–addmrna Add ‘mRNA’ features for each ‘CDS’ feature (default OFF)
–locustag [X] Locus tag prefix [auto] (default ‘’)
–increment [N] Locus tag counter increment (default ‘1’)
–gffver [N] GFF version (default ‘3’)
–compliant Force Genbank/ENA/DDJB compliance: --addgenes --mincontiglen 200 --centre XXX (default OFF)
–centre [X] Sequencing centre ID. (default ‘’)
–accver [N] Version to put in Genbank file (default ‘1’)
Organism details:
–genus [X] Genus name (default ‘Genus’)
–species [X] Species name (default ‘species’)
–strain [X] Strain name (default ‘strain’)
–plasmid [X] Plasmid name or identifier (default ‘’)
Annotations:
–kingdom [X] Annotation mode: Archaea|Bacteria|Mitochondria|Viruses (default ‘Bacteria’)
–gcode [N] Genetic code / Translation table (set if --kingdom is set) (default ‘0’)
–gram [X] Gram: -/neg +/pos (default ‘’)
–usegenus Use genus-specific BLAST databases (needs --genus) (default OFF)
–proteins [X] FASTA or GBK file to use as 1st priority (default ‘’)
–hmms [X] Trusted HMM to first annotate from (default ‘’)
–metagenome Improve gene predictions for highly fragmented genomes (default OFF)
–rawproduct Do not clean up /product annotation (default OFF)
–cdsrnaolap Allow [tr]RNA to overlap CDS (default OFF)
Computation:
–cpus [N] Number of CPUs to use [0=all] (default ‘8’)
–fast Fast mode - only use basic BLASTP databases (default OFF)
–noanno For CDS just set /product=“unannotated protein” (default OFF)
–mincontiglen [N] Minimum contig size [NCBI needs 200] (default ‘1’)
–evalue [n.n] Similarity e-value cut-off (default ‘1e-06’)
–rfam Enable searching for ncRNAs with Infernal+Rfam (SLOW!) (default ‘0’)
–norrna Don’t run rRNA search (default OFF)
–notrna Don’t run tRNA search (default OFF)
–rnammer Prefer RNAmmer over Barrnap for rRNA prediction (default OFF)

‘nice make -j 1 -C /home/ubuntu/efn/concat_reads/efn_nullarbor_run03Apr2018’ is currently running, I will share the output once it’s done please.

Thx
Ebenn

[Ebenn]

Here is the output from ‘nice make -j 1 -C /home/ubuntu/efn/concat_reads/efn_nullarbor_run03Apr2018’ please:

ubuntu@ebenn:~/efn$ prokka --outdir UMN_new_prokka --prefix Eco reference_new_EcoliUMN206.fasta
[11:37:29] This is prokka 1.12
[11:37:29] Written by Torsten Seemann torsten.seemann@gmail.com
[11:37:29] Homepage is https://github.com/tseemann/prokka
[11:37:29] Local time is Wed Apr 4 11:37:29 2018
[11:37:29] You are ubuntu
[11:37:29] Operating system is linux
[11:37:29] You have BioPerl 1.006924
[11:37:29] System has 4 cores.
[11:37:29] Option --cpu asked for 8 cores, but system only has 4
[11:37:29] Will use maximum of 4 cores.
[11:37:29] Annotating as >>> Bacteria <<<
[11:37:29] Generating locus_tag from ‘reference_new_EcoliUMN206.fasta’ contents.
[11:37:29] Setting --locustag OMINJGOG from MD5 862730805f5650c5bbf6d772493f3967
[11:37:29] Creating new output folder: UMN_new_prokka
[11:37:29] Running: mkdir -p UMN_new_prokka
[11:37:29] Using filename prefix: Eco.XXX
[11:37:29] Setting HMMER_NCPU=1
[11:37:29] Writing log to: UMN_new_prokka/Eco.log
[11:37:29] Command: /home/linuxbrew/.linuxbrew/bin/prokka --outdir UMN_new_prokka --prefix Eco reference_new_EcoliUMN206.fasta
[11:37:29] Appending to PATH: /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/binaries/linux
[11:37:29] Appending to PATH: /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/binaries/linux/…/common
[11:37:29] Appending to PATH: /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin
[11:37:29] Looking for ‘aragorn’ - found /home/linuxbrew/.linuxbrew/bin/aragorn
[11:37:29] Determined aragorn version is 1.2
[11:37:29] Looking for ‘barrnap’ - found /home/linuxbrew/.linuxbrew/bin/barrnap
[11:37:29] Determined barrnap version is 0.8
[11:37:29] Looking for ‘blastp’ - found /home/linuxbrew/.linuxbrew/bin/blastp
[11:37:29] Determined blastp version is 2.6
[11:37:29] Looking for ‘cmpress’ - found /home/linuxbrew/.linuxbrew/bin/cmpress
[11:37:29] Determined cmpress version is 1.1
[11:37:29] Looking for ‘cmscan’ - found /home/linuxbrew/.linuxbrew/bin/cmscan
[11:37:29] Determined cmscan version is 1.1
[11:37:29] Looking for ‘egrep’ - found /bin/egrep
[11:37:29] Looking for ‘find’ - found /usr/bin/find
[11:37:29] Looking for ‘grep’ - found /bin/grep
[11:37:29] Looking for ‘hmmpress’ - found /home/linuxbrew/.linuxbrew/bin/hmmpress
[11:37:29] Determined hmmpress version is 3.1
[11:37:29] Looking for ‘hmmscan’ - found /home/linuxbrew/.linuxbrew/bin/hmmscan
[11:37:29] Determined hmmscan version is 3.1
[11:37:29] Looking for ‘java’ - found /home/linuxbrew/.linuxbrew/bin/java
[11:37:29] Looking for ‘less’ - found /usr/bin/less
[11:37:29] Looking for ‘makeblastdb’ - found /home/linuxbrew/.linuxbrew/bin/makeblastdb
[11:37:29] Determined makeblastdb version is 2.6
[11:37:29] Looking for ‘minced’ - found /home/linuxbrew/.linuxbrew/bin/minced
[11:37:30] Determined minced version is 2.0
[11:37:30] Looking for ‘parallel’ - found /home/linuxbrew/.linuxbrew/bin/parallel
[11:37:30] Determined parallel version is 20170922
[11:37:30] Looking for ‘prodigal’ - found /home/linuxbrew/.linuxbrew/bin/prodigal
[11:37:30] Determined prodigal version is 2.6
[11:37:30] Looking for ‘prokka-genbank_to_fasta_db’ - found /home/linuxbrew/.linuxbrew/bin/prokka-genbank_to_fasta_db
[11:37:30] Looking for ‘sed’ - found /bin/sed
[11:37:30] Looking for ‘tbl2asn’ - found /home/linuxbrew/.linuxbrew/bin/tbl2asn
[tbl2asn] This copy of tbl2asn is more than a year old. Please download the current version.
[11:37:30] Determined tbl2asn version is 25.3
[11:37:30] Using genetic code table 11.
[11:37:30] Loading and checking input file: reference_new_EcoliUMN206.fasta
[11:37:30] Wrote 1 contigs totalling 5202090 bp.
[11:37:30] Predicting tRNAs and tmRNAs
[11:37:30] Running: aragorn -l -gc11 -w UMN_new_prokka/Eco.fna
[11:37:34] 1 tRNA-Ile [228231,228307] 35 (gat)
[11:37:34] 2 tRNA-Ala [228350,228425] 34 (tgc)
[11:37:34] 3 tRNA-Asp [231779,231855] 35 (gtc)
[11:37:34] 4 tRNA-Asp [240002,240078] 35 (gtc)
[11:37:34] 5 tRNA-Thr [335532,335607] 34 (cgt)
[11:37:34] 6 tRNA-Ser [403247,403327] 38 (gga)
[11:37:34] 7 tRNA-Arg [644851,644927] 35 (tct)
[11:37:34] 8 tRNA-Gln c[808736,808810] 33 (ctg)
[11:37:34] 9 tRNA-Gln c[808848,808922] 33 (ctg)
[11:37:34] 10 tRNA-Met c[808971,809047] 35 (cat)
[11:37:34] 11 tRNA-Gln c[809063,809137] 33 (ttg)
[11:37:34] 12 tRNA-Gln c[809172,809246] 33 (ttg)
[11:37:34] 13 tRNA-Leu c[809270,809354] 35 (tag)
[11:37:34] 14 tRNA-Met c[809364,809440] 35 (cat)
[11:37:34] 15 tRNA-Lys [888195,888270] 34 (ttt)
[11:37:34] 16 tRNA-Val [888406,888481] 34 (tac)
[11:37:34] 17 tRNA-Lys [888484,888559] 34 (ttt)
[11:37:34] 18 tRNA-Val [888611,888686] 34 (tac)
[11:37:34] 19 tRNA-Lys [888690,888765] 34 (ttt)
[11:37:34] 20 tRNA-Lys [888912,888987] 34 (ttt)
[11:37:34] 21 tRNA-Lys [889021,889096] 34 (ttt)
[11:37:34] 22 tRNA-Gly [980827,980911] 37 (gcc)
[11:37:34] 23 tRNA-Ser c[1115007,1115094] 35 (gga)
[11:37:34] 24 tRNA-Ser c[1213806,1213893] 35 (tga)
[11:37:34] 25 tRNA-Ser c[1262465,1262552] 35 (gga)
[11:37:34] 26 tRNA-Tyr c[1540737,1540821] 35 (gta)
[11:37:34] 27 tRNA-Tyr c[1540856,1540940] 35 (gta)
[11:37:34] 28 tRNA-Val [1981213,1981289] 35 (gac)
[11:37:34] 29 tRNA-Val [1981294,1981370] 35 (gac)
[11:37:34] 30 tRNA-Leu c[2225250,2225336] 35 (taa)
[11:37:34] 31 tRNA-Cys c[2225349,2225422] 33 (gca)
[11:37:34] 32 tRNA-Gly c[2225477,2225552] 34 (gcc)
[11:37:34] 33 tRNA-Ser c[2276274,2276363] 35 (cga)
[11:37:34] 34 tRNA-Asn [2277355,2277430] 34 (gtt)
[11:37:34] 35 tRNA-Asn c[2357471,2357546] 34 (gtt)
[11:37:34] 36 tRNA-Asn [2359295,2359370] 34 (gtt)
[11:37:34] 37 tRNA-Asn [2361703,2361778] 34 (gtt)
[11:37:34] 38 tRNA-Pro [2588625,2588701] 35 (ggg)
[11:37:34] 39 tRNA-Arg [2765909,2765983] 34 (cct)
[11:37:34] 40 tRNA-Ala c[2809275,2809350] 34 (ggc)
[11:37:34] 41 tRNA-Ala c[2809390,2809465] 34 (ggc)
[11:37:34] 42 tRNA-Val [2812164,2812239] 34 (tac)
[11:37:34] 43 tRNA-Val [2812284,2812359] 34 (tac)
[11:37:34] 44 tRNA-Val [2812406,2812481] 34 (tac)
[11:37:34] 45 tRNA-Lys [2812486,2812561] 34 (ttt)
[11:37:34] 46 tRNA-Glu c[3022365,3022440] 35 (ttc)
[11:37:34] 47 tmRNA [3048497,3048859] 90,125 ANDENYALAA**
[11:37:34] 48 tRNA-Met c[3089116,3089191] 34 (cat)
[11:37:34] 49 tRNA-Arg c[3123350,3123426] 35 (acg)
[11:37:34] 50 tRNA-Arg c[3123625,3123701] 35 (acg)
[11:37:34] 51 tRNA-Arg c[3123899,3123975] 35 (acg)
[11:37:34] 52 tRNA-Arg [3124159,3124230] 34 (tcg)
[11:37:34] 53 tRNA-Arg c[3124174,3124250] 35 (acg)
[11:37:34] 54 tRNA-Ser c[3124254,3124346] 35 (gct)
[11:37:34] 55 tRNA-Met [3253901,3253977] 35 (cat)
[11:37:34] 56 tRNA-Met [3254011,3254087] 35 (cat)
[11:37:34] 57 tRNA-Met [3254120,3254196] 35 (cat)
[11:37:34] 58 tRNA-Met [3254229,3254305] 35 (cat)
[11:37:34] 59 tRNA-Gly c[3321545,3321618] 33 (ccc)
[11:37:34] 60 tRNA-Phe [3445924,3445999] 34 (gaa)
[11:37:34] 61 tRNA-Met [3670374,3670449] 34 (cat)
[11:37:34] 62 tRNA-Met c[3771415,3771491] 35 (cat)
[11:37:34] 63 tRNA-Leu c[3775274,3775360] 35 (gag)
[11:37:34] 64 tRNA-Thr c[3869830,3869905] 34 (ggt)
[11:37:34] 65 tRNA-Ala c[3873219,3873294] 34 (tgc)
[11:37:34] 66 tRNA-Ile c[3873337,3873413] 35 (gat)
[11:37:34] 67 tRNA-Pro c[4171557,4171633] 35 (cgg)
[11:37:34] 68 tRNA-seC [4296805,4296899] 35 (tca)
[11:37:34] 69 tRNA-Glu [4430302,4430377] 35 (ttc)
[11:37:34] 70 tRNA-Asp [4433738,4433814] 35 (gtc)
[11:37:34] 71 tRNA-Trp [4433823,4433898] 34 (cca)
[11:37:34] 72 tRNA-Arg [4468367,4468443] 35 (ccg)
[11:37:34] 73 tRNA-His [4468502,4468577] 34 (gtg)
[11:37:34] 74 tRNA-Ser [4468598,4468684] 35 (cag)
[11:37:34] 75 tRNA-Pro [4468727,4468803] 35 (tgg)
[11:37:34] 76 tRNA-Ile [4523799,4523875] 35 (gat)
[11:37:34] 77 tRNA-Ala [4523918,4523993] 34 (tgc)
[11:37:34] 78 tRNA-Glu [4657672,4657747] 35 (ttc)
[11:37:34] 79 tRNA-Thr [4664525,4664600] 34 (tgt)
[11:37:34] 80 tRNA-Tyr [4664609,4664693] 35 (gta)
[11:37:34] 81 tRNA-Gly [4664810,4664884] 34 (tcc)
[11:37:34] 82 tRNA-Thr [4664891,4664966] 34 (ggt)
[11:37:34] 83 tRNA-Glu [4698766,4698841] 35 (ttc)
[11:37:34] 84 tRNA-Phe c[4848121,4848196] 34 (gaa)
[11:37:34] 85 tRNA-Gly [4877050,4877125] 34 (gcc)
[11:37:34] 86 tRNA-Gly [4877283,4877358] 34 (gcc)
[11:37:34] 87 tRNA-Gly [4877394,4877469] 34 (gcc)
[11:37:34] 88 tRNA-Leu [4983917,4984001] 35 (caa)
[11:37:34] 89 tRNA-Ser c[5163404,5163490] 35 (cag)
[11:37:34] 90 tRNA-Ser c[5163525,5163611] 35 (cag)
[11:37:34] 91 tRNA-Ser c[5163640,5163726] 35 (cag)
[11:37:34] Found 91 tRNAs
[11:37:34] Predicting Ribosomal RNAs
[11:37:34] Running Barrnap with 4 threads
[11:37:39] 1 CU928163 226624 16S ribosomal RNA
[11:37:39] 2 CU928163 228611 23S ribosomal RNA
[11:37:39] 3 CU928163 231611 5S ribosomal RNA
[11:37:39] 4 CU928163 3019070 5S ribosomal RNA
[11:37:39] 5 CU928163 3019277 23S ribosomal RNA
[11:37:39] 6 CU928163 3022527 16S ribosomal RNA
[11:37:39] 7 CU928163 3869678 5S ribosomal RNA
[11:37:39] 8 CU928163 3869923 5S ribosomal RNA
[11:37:39] 9 CU928163 3870132 23S ribosomal RNA
[11:37:39] 10 CU928163 3873483 16S ribosomal RNA
[11:37:39] 11 CU928163 4428678 16S ribosomal RNA
[11:37:39] 12 CU928163 4430573 23S ribosomal RNA
[11:37:39] 13 CU928163 4433570 5S ribosomal RNA
[11:37:39] 14 CU928163 4522192 16S ribosomal RNA
[11:37:39] 15 CU928163 4524179 23S ribosomal RNA
[11:37:39] 16 CU928163 4527174 5S ribosomal RNA
[11:37:39] 17 CU928163 4656048 16S ribosomal RNA
[11:37:39] 18 CU928163 4657943 23S ribosomal RNA
[11:37:39] 19 CU928163 4660944 5S ribosomal RNA
[11:37:39] 20 CU928163 4697142 16S ribosomal RNA
[11:37:39] 21 CU928163 4699037 23S ribosomal RNA
[11:37:39] 22 CU928163 4702038 5S ribosomal RNA
[11:37:39] Found 22 rRNAs
[11:37:39] Skipping ncRNA search, enable with --rfam if desired.
[11:37:39] Total of 112 tRNA + rRNA features
[11:37:39] Searching for CRISPR repeats
[11:37:40] CRISPR1 CU928163 3181533 with 18 spacers
[11:37:40] CRISPR2 CU928163 3208301 with 10 spacers
[11:37:40] Found 2 CRISPRs
[11:37:40] Predicting coding sequences
[11:37:40] Contigs total 5202090 bp, so using single mode
[11:37:40] Running: prodigal -i UMN_new_prokka/Eco.fna -c -m -g 11 -p single -f sco -q
[11:37:53] Excluding CDS which overlaps existing RNA (rRNA) at CU928163:227808…228062 on + strand
[11:37:55] Excluding CDS which overlaps existing RNA (tRNA) at CU928163:978946…981231 on + strand
[11:37:57] Excluding CDS which overlaps existing RNA (tRNA) at CU928163:1540558…1541004 on + strand
[11:37:59] Excluding CDS which overlaps existing RNA (tRNA) at CU928163:2588655…2588780 on - strand
[11:38:00] Excluding CDS which overlaps existing RNA (rRNA) at CU928163:3022626…3022880 on - strand
[11:38:02] Excluding CDS which overlaps existing RNA (tRNA) at CU928163:3869753…3870280 on + strand
[11:38:02] Excluding CDS which overlaps existing RNA (rRNA) at CU928163:3873582…3873836 on - strand
[11:38:04] Excluding CDS which overlaps existing RNA (rRNA) at CU928163:4429862…4430116 on + strand
[11:38:04] Excluding CDS which overlaps existing RNA (rRNA) at CU928163:4523376…4523630 on + strand
[11:38:04] Excluding CDS which overlaps existing RNA (rRNA) at CU928163:4657232…4657486 on + strand
[11:38:04] Excluding CDS which overlaps existing RNA (rRNA) at CU928163:4698326…4698580 on + strand
[11:38:06] Found 4814 CDS
[11:38:06] Connecting features back to sequences
[11:38:06] Not using genus-specific database. Try --usegenus to enable it.
[11:38:06] Annotating CDS, please be patient.
[11:38:06] Will use 4 CPUs for similarity searching.
[11:38:12] There are still 4814 unannotated CDS left (started with 4814)
[11:38:12] Will use blast to search against /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/db/kingdom/Bacteria/sprot with 4 CPUs
[11:38:12] Running: cat UMN_new_prokka/sprot.faa | parallel --gnu --plain -j 4 --block 194211 --recstart ‘>’ --pipe blastp -query - -db /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/db/kingdom/Bacteria/sprot -evalue 1e-06 -num_threads 1 -num_descriptions 1 -num_alignments 1 -seg no > UMN_new_prokka/sprot.blast 2> /dev/null
[11:40:26] Modify product: Uncharacterized metal-dependent hydrolase YcfH => putative metal-dependent hydrolase YcfH
[11:40:26] Modify product: Probable L,D-transpeptidase YcfS => putative L,D-transpeptidase YcfS
[11:40:26] Modify product: Prophage tail fiber assembly protein homolog TfaE => Prophage tail fiber assembly protein TfaE
[11:40:26] Modify product: Uncharacterized ABC transporter ATP-binding protein HI_1470 => putative ABC transporter ATP-binding protein
[11:40:26] Modify product: Probable two-component-system connector protein YcgZ => putative two-component-system connector protein YcgZ
[11:40:26] Modify product: Probable two-component-system connector protein AriR => putative two-component-system connector protein AriR
[11:40:26] Modify product: Probable autotransporter ROD_p1121 => putative autotransporter
[11:40:26] Modify product: Uncharacterized ABC transporter ATP-binding protein HI_1470 => putative ABC transporter ATP-binding protein
[11:40:26] Modify product: Probable ABC transporter permease protein HI_1471 => putative ABC transporter permease protein
[11:40:26] Modify product: Probable autotransporter ROD_p1121 => putative autotransporter
[11:40:26] Modify product: Uncharacterized NTE family protein Rv1063c => putative NTE family protein
[11:40:26] Modify product: Uncharacterized acyl-CoA thioester hydrolase HI_0827 => putative acyl-CoA thioester hydrolase
[11:40:26] Modify product: Probable intracellular septation protein A => putative intracellular septation protein A
[11:40:26] Modify product: Uncharacterized oxidoreductase YciK => putative oxidoreductase YciK
[11:40:26] Modify product: Probable protease SohB => putative protease SohB

…‘modify product …’ continues…

[11:40:34] Modify product: Uncharacterized metal-dependent hydrolase YjjV => putative metal-dependent hydrolase YjjV
[11:40:34] Cleaned 240 /product names
[11:40:34] Deleting unwanted file: UMN_new_prokka/sprot.faa
[11:40:34] Deleting unwanted file: UMN_new_prokka/sprot.blast
[11:40:36] There are still 1347 unannotated CDS left (started with 4814)
[11:40:36] Will use hmmer3 to search against /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/db/hmm/HAMAP.hmm with 4 CPUs
[11:40:36] Running: cat UMN_new_prokka/HAMAP.hmm.faa | parallel --gnu --plain -j 4 --block 40697 --recstart ‘>’ --pipe hmmscan --noali --notextw --acc -E 1e-06 --cpu 1 /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/db/hmm/HAMAP.hmm /dev/stdin > UMN_new_prokka/HAMAP.hmm.hmmer3 2> /dev/null
[11:41:30] Modify product: Uncharacterized Nudix hydrolase NudL => putative Nudix hydrolase NudL
[11:41:31] Cleaned 1 /product names
[11:41:31] Deleting unwanted file: UMN_new_prokka/HAMAP.hmm.faa
[11:41:31] Deleting unwanted file: UMN_new_prokka/HAMAP.hmm.hmmer3
[11:41:31] Labelling remaining 1269 proteins as ‘hypothetical protein’
[11:41:31] Possible /pseudo ‘HTH-type transcriptional regulator PgrR’ at CU928163 position 360996
[11:41:31] Possible /pseudo ‘Manganese transport system membrane protein MntB’ at CU928163 position 1453162
[11:41:31] Possible /pseudo ‘Small toxic polypeptide LdrD’ at CU928163 position 1524026
[11:41:31] Possible /pseudo ‘Formate dehydrogenase, nitrate-inducible, major subunit’ at CU928163 position 1745854
[11:41:31] Possible /pseudo ‘Aldehyde-alcohol dehydrogenase’ at CU928163 position 2379424
[11:41:31] Possible /pseudo ‘L-aspartate oxidase’ at CU928163 position 3002920
[11:41:31] Possible /pseudo ‘Energy-coupling factor transporter ATP-binding protein EcfA1’ at CU928163 position 3402066
[11:41:31] Possible /pseudo ‘Phosphoethanolamine transferase OpgE’ at CU928163 position 3449574
[11:41:31] Possible /pseudo ‘putative FAD-linked oxidoreductase’ at CU928163 position 3576245
[11:41:31] Possible /pseudo ‘Acyl carrier protein’ at CU928163 position 4049865
[11:41:31] Possible /pseudo ‘Cellulose synthase operon protein C’ at CU928163 position 4149324
[11:41:31] Possible /pseudo ‘Cellulose synthase catalytic subunit [UDP-forming]’ at CU928163 position 4156313
[11:41:31] Possible /pseudo ‘Small toxic polypeptide LdrD’ at CU928163 position 4161808
[11:41:31] Possible /pseudo ‘Adenosyl-chloride synthase’ at CU928163 position 4486626
[11:41:31] Possible /pseudo ‘Putative 2,3-dihydroxypropane-1-sulfonate exporter’ at CU928163 position 4553402
[11:41:31] Possible /pseudo ‘Formate dehydrogenase-O major subunit’ at CU928163 position 4574689
[11:41:31] Possible /pseudo ‘Formate dehydrogenase H’ at CU928163 position 4791797
[11:41:31] Possible /pseudo ‘putative L-galactonate transporter’ at CU928163 position 5151209
[11:41:31] Found 3021 unique /gene codes.
[11:41:31] Fixed 3 duplicate /gene - esiB_1 esiB_2 esiB_3
[11:41:31] Fixed 2 duplicate /gene - nagA_1 nagA_2
[11:41:31] Fixed 2 duplicate /gene - arcC2_1 arcC2_2
[11:41:31] Fixed 2 duplicate /gene - nanA_1 nanA_2
[11:41:31] Fixed 2 duplicate /gene - glrR_1 glrR_2
[11:41:31] Fixed 4 duplicate /gene - ldrD_1 ldrD_2 ldrD_3 ldrD_4
[11:41:31] Fixed 2 duplicate /gene - srlB_1 srlB_2
[11:41:31] Fixed 2 duplicate /gene - epsE_1 epsE_2
[11:41:31] Fixed 2 duplicate /gene - lgoT_1 lgoT_2
[11:41:31] Fixed 3 duplicate /gene - srmB_1 srmB_2 srmB_3
[11:41:31] Fixed 3 duplicate /gene - gltD_1 gltD_2 gltD_3
[11:41:31] Fixed 2 duplicate /gene - flgF_1 flgF_2

…‘Fixed 2 duplicate…’ continues…

[11:41:31] Adding /locus_tag identifiers
[11:41:31] Assigned 4927 locus_tags to CDS and RNA features.
[11:41:31] Writing outputs to UMN_new_prokka/
[11:41:40] Generating annotation statistics file
[11:41:40] Generating Genbank and Sequin files
[11:41:40] Running: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y ‘Annotated using prokka 1.12 from https://github.com/tseemann/prokka’ -Z UMN_new_prokka/Eco.err -i UMN_new_prokka/Eco.fsa 2> /dev/null
[11:41:54] Could not run command: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y ‘Annotated using prokka 1.12 from https://github.com/tseemann/prokka’ -Z UMN_new_prokka/Eco.err -i UMN_new_prokka/Eco.fsa 2> /dev/null
ubuntu@ebenn:~/efn$ prokka
Name:
Prokka 1.12 by Torsten Seemann torsten.seemann@gmail.com
Synopsis:
rapid bacterial genome annotation
Usage:
prokka [options] <contigs.fasta>
General:
–help This help
–version Print version and exit
–docs Show full manual/documentation
–citation Print citation for referencing Prokka
–quiet No screen output (default OFF)
–debug Debug mode: keep all temporary files (default OFF)
Setup:
–listdb List all configured databases
–setupdb Index all installed databases
–cleandb Remove all database indices
–depends List all software dependencies
Outputs:
–outdir [X] Output folder [auto] (default ‘’)
–force Force overwriting existing output folder (default OFF)
–prefix [X] Filename output prefix [auto] (default ‘’)
–addgenes Add ‘gene’ features for each ‘CDS’ feature (default OFF)
–addmrna Add ‘mRNA’ features for each ‘CDS’ feature (default OFF)
–locustag [X] Locus tag prefix [auto] (default ‘’)
–increment [N] Locus tag counter increment (default ‘1’)
–gffver [N] GFF version (default ‘3’)
–compliant Force Genbank/ENA/DDJB compliance: --addgenes --mincontiglen 200 --centre XXX (default OFF)
–centre [X] Sequencing centre ID. (default ‘’)
–accver [N] Version to put in Genbank file (default ‘1’)
Organism details:
–genus [X] Genus name (default ‘Genus’)
–species [X] Species name (default ‘species’)
–strain [X] Strain name (default ‘strain’)
–plasmid [X] Plasmid name or identifier (default ‘’)
Annotations:
–kingdom [X] Annotation mode: Archaea|Bacteria|Mitochondria|Viruses (default ‘Bacteria’)
–gcode [N] Genetic code / Translation table (set if --kingdom is set) (default ‘0’)
–gram [X] Gram: -/neg +/pos (default ‘’)
–usegenus Use genus-specific BLAST databases (needs --genus) (default OFF)
–proteins [X] FASTA or GBK file to use as 1st priority (default ‘’)
–hmms [X] Trusted HMM to first annotate from (default ‘’)
–metagenome Improve gene predictions for highly fragmented genomes (default OFF)
–rawproduct Do not clean up /product annotation (default OFF)
–cdsrnaolap Allow [tr]RNA to overlap CDS (default OFF)
Computation:
–cpus [N] Number of CPUs to use [0=all] (default ‘8’)
–fast Fast mode - only use basic BLASTP databases (default OFF)
–noanno For CDS just set /product=“unannotated protein” (default OFF)
–mincontiglen [N] Minimum contig size [NCBI needs 200] (default ‘1’)
–evalue [n.n] Similarity e-value cut-off (default ‘1e-06’)
–rfam Enable searching for ncRNAs with Infernal+Rfam (SLOW!) (default ‘0’)
–norrna Don’t run rRNA search (default OFF)
–notrna Don’t run tRNA search (default OFF)
–rnammer Prefer RNAmmer over Barrnap for rRNA prediction (default OFF)
ubuntu@ebenn:~/efn$ nice make -j 1 -C /home/ubuntu/efn/concat_reads/efn_nullarbor_run03Apr2018
make: Entering directory ‘/home/ubuntu/efn/concat_reads/efn_nullarbor_run03Apr2018’
mkdir -p sample1
mkdir -p sample2
mkdir -p sample3
mkdir -p sample4
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929580-1_S95_L999_R1_001.fastq.gz’ ‘sample1/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929580-1_S95_L999_R2_001.fastq.gz’ ‘sample1/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929583-1_S83_L999_R1_001.fastq.gz’ ‘sample2/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929583-1_S83_L999_R2_001.fastq.gz’ ‘sample2/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-1_S80_L999_R1_001.fastq.gz’ ‘sample3/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-1_S80_L999_R2_001.fastq.gz’ ‘sample3/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929580-10_S68_L999_R1_001.fastq.gz’ ‘sample4/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929580-10_S68_L999_R2_001.fastq.gz’ ‘sample4/R2.fq.gz’
prokka --centre X --compliant --force --fast --gcode 0 --locustag sample1 --prefix sample1 --outdir sample1/prokka --cpus 4 sample1/contigs.fa
[11:43:37] This is prokka 1.12
[11:43:37] Written by Torsten Seemann torsten.seemann@gmail.com
[11:43:37] Homepage is https://github.com/tseemann/prokka
[11:43:37] Local time is Wed Apr 4 11:43:37 2018
[11:43:37] You are ubuntu
[11:43:37] Operating system is linux
[11:43:37] You have BioPerl 1.006924
[11:43:37] System has 4 cores.
[11:43:37] Will use maximum of 4 cores.
[11:43:37] Annotating as >>> Bacteria <<<
[11:43:37] Enabling options to ensure Genbank/ENA/DDJB submission compliance.
[11:43:37] Creating new output folder: sample1/prokka
[11:43:37] Running: mkdir -p sample1/prokka
[11:43:37] Using filename prefix: sample1.XXX
[11:43:37] Setting HMMER_NCPU=1
[11:43:37] Writing log to: sample1/prokka/sample1.log
[11:43:37] Command: /home/linuxbrew/.linuxbrew/bin/prokka --centre X --compliant --force --fast --gcode 0 --locustag sample1 --prefix sample1 --outdir sample1/prokka --cpus 4 sample1/contigs.fa
[11:43:37] Appending to PATH: /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/binaries/linux
[11:43:37] Appending to PATH: /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/binaries/linux/…/common
[11:43:37] Appending to PATH: /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin
[11:43:37] Looking for ‘aragorn’ - found /home/linuxbrew/.linuxbrew/bin/aragorn
[11:43:37] Determined aragorn version is 1.2
[11:43:37] Looking for ‘barrnap’ - found /home/linuxbrew/.linuxbrew/bin/barrnap
[11:43:37] Determined barrnap version is 0.8
[11:43:37] Looking for ‘blastp’ - found /home/linuxbrew/.linuxbrew/bin/blastp
[11:43:37] Determined blastp version is 2.6
[11:43:37] Looking for ‘cmpress’ - found /home/linuxbrew/.linuxbrew/bin/cmpress
[11:43:37] Determined cmpress version is 1.1
[11:43:37] Looking for ‘cmscan’ - found /home/linuxbrew/.linuxbrew/bin/cmscan
[11:43:37] Determined cmscan version is 1.1
[11:43:37] Looking for ‘egrep’ - found /bin/egrep
[11:43:37] Looking for ‘find’ - found /usr/bin/find
[11:43:37] Looking for ‘grep’ - found /bin/grep
[11:43:37] Looking for ‘hmmpress’ - found /home/linuxbrew/.linuxbrew/bin/hmmpress
[11:43:37] Determined hmmpress version is 3.1
[11:43:37] Looking for ‘hmmscan’ - found /home/linuxbrew/.linuxbrew/bin/hmmscan
[11:43:37] Determined hmmscan version is 3.1
[11:43:37] Looking for ‘java’ - found /home/linuxbrew/.linuxbrew/bin/java
[11:43:37] Looking for ‘less’ - found /usr/bin/less
[11:43:37] Looking for ‘makeblastdb’ - found /home/linuxbrew/.linuxbrew/bin/makeblastdb
[11:43:37] Determined makeblastdb version is 2.6
[11:43:37] Looking for ‘minced’ - found /home/linuxbrew/.linuxbrew/bin/minced
[11:43:37] Determined minced version is 2.0
[11:43:37] Looking for ‘parallel’ - found /home/linuxbrew/.linuxbrew/bin/parallel
[11:43:38] Determined parallel version is 20170922
[11:43:38] Looking for ‘prodigal’ - found /home/linuxbrew/.linuxbrew/bin/prodigal
[11:43:38] Determined prodigal version is 2.6
[11:43:38] Looking for ‘prokka-genbank_to_fasta_db’ - found /home/linuxbrew/.linuxbrew/bin/prokka-genbank_to_fasta_db
[11:43:38] Looking for ‘sed’ - found /bin/sed
[11:43:38] Looking for ‘tbl2asn’ - found /home/linuxbrew/.linuxbrew/bin/tbl2asn
[tbl2asn] This copy of tbl2asn is more than a year old. Please download the current version.
[11:43:38] Determined tbl2asn version is 25.3
[11:43:38] Using genetic code table 11.
[11:43:38] Loading and checking input file: sample1/contigs.fa
[11:43:39] Wrote 3310 contigs totalling 7431003 bp.
[11:43:39] Predicting tRNAs and tmRNAs
[11:43:39] Running: aragorn -l -gc11 -w sample1/prokka/sample1.fna
[11:43:39] 1 tRNA-Leu [152,236] 35 (caa)
[11:43:39] 1 tRNA-seC [1380,1474] 35 (tca)
[11:43:39] 1 tRNA-Thr [1478,1553] 34 (tgt)
[11:43:39] 2 tRNA-Tyr [1562,1646] 35 (gta)
[11:43:39] 3 tRNA-Gly [1763,1837] 34 (tcc)
[11:43:39] 4 tRNA-Thr [1844,1919] 34 (ggt)
[11:43:39] 1 tRNA-Val c[1867,1943] 35 (gac)

…‘list of tRNA…’ continues…

[11:43:44] Found 95 tRNAs
[11:43:44] Predicting Ribosomal RNAs
[11:43:44] Running Barrnap with 4 threads
[11:43:47] 1 gnl|X|sample1_1070 638 5S ribosomal RNA
[11:43:47] 2 gnl|X|sample1_1070 883 5S ribosomal RNA
[11:43:47] 3 gnl|X|sample1_1192 4 5S ribosomal RNA
[11:43:47] 4 gnl|X|sample1_1349 5 16S ribosomal RNA
[11:43:47] 5 gnl|X|sample1_1800 2 23S ribosomal RNA (partial)
[11:43:47] 6 gnl|X|sample1_782 198 5S ribosomal RNA
[11:43:47] 7 gnl|X|sample1_914 687 5S ribosomal RNA
[11:43:47] Found 7 rRNAs
[11:43:47] Skipping ncRNA search, enable with --rfam if desired.
[11:43:47] Total of 102 tRNA + rRNA features
[11:43:47] Searching for CRISPR repeats
[11:43:47] CRISPR1 gnl|X|sample1_1799 557 with 10 spacers
[11:43:48] CRISPR2 gnl|X|sample1_2500 123 with 17 spacers
[11:43:48] Found 2 CRISPRs
[11:43:48] Predicting coding sequences
[11:43:48] Contigs total 7431003 bp, so using single mode
[11:43:48] Running: prodigal -i sample1/prokka/sample1.fna -c -m -g 11 -p single -f sco -q
[11:43:58] Excluding CDS which overlaps existing RNA (tRNA) at gnl|X|sample1_20:31…297 on - strand
[11:43:59] Excluding CDS which overlaps existing RNA (tRNA) at gnl|X|sample1_876:88…423 on + strand
[11:44:00] Excluding CDS which overlaps existing RNA (tRNA) at gnl|X|sample1_1070:713…985 on + strand
[11:44:00] Excluding CDS which overlaps existing RNA (rRNA) at gnl|X|sample1_1349:1051…1305 on + strand
[11:44:00] Excluding CDS which overlaps existing RNA (tRNA) at gnl|X|sample1_1639:15934…16185 on - strand
[11:44:01] Excluding CDS which overlaps existing RNA (repeat_region) at gnl|X|sample1_2500:519…1034 on + strand
[11:44:02] Excluding CDS which overlaps existing RNA (tRNA) at gnl|X|sample1_3137:16280…17653 on + strand
[11:44:02] Found 7122 CDS
[11:44:02] Connecting features back to sequences
[11:44:02] Not using genus-specific database. Try --usegenus to enable it.
[11:44:02] Annotating CDS, please be patient.
[11:44:02] Will use 4 CPUs for similarity searching.
[11:44:06] There are still 7122 unannotated CDS left (started with 7122)
[11:44:06] Will use blast to search against /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/db/kingdom/Bacteria/sprot with 4 CPUs
[11:44:06] Running: cat sample1/prokka/sprot.faa | parallel --gnu --plain -j 4 --block 220968 --recstart ‘>’ --pipe blastp -query - -db /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/db/kingdom/Bacteria/sprot -evalue 1e-06 -num_threads 1 -num_descriptions 1 -num_alignments 1 -seg no > sample1/prokka/sprot.blast 2> /dev/null
[11:46:35] Modify product: Uncharacterized HTH-type transcriptional regulator YbbH => putative HTH-type transcriptional regulator YbbH

… (truncating ‘modify product’ output to save space)…

[11:46:47] Modify product: Cytochrome b561 homolog 1 => Cytochrome b561
[11:46:47] Cleaned 306 /product names
[11:46:47] Deleting unwanted file: sample1/prokka/sprot.faa
[11:46:47] Deleting unwanted file: sample1/prokka/sprot.blast
[11:46:47] In --fast mode so skipping non-BLAST search against /home/linuxbrew/.linuxbrew/Cellar/prokka/HEAD-f7f819b/bin/…/db/hmm/HAMAP.hmm
[11:46:47] Labelling remaining 2692 proteins as ‘hypothetical protein’
[11:46:47] Possible /pseudo ‘Putative 2,3-dihydroxypropane-1-sulfonate exporter’ at gnl|X|sample1_50 position 10749
[11:46:47] Possible /pseudo ‘Membrane-associated protein UidC’ at gnl|X|sample1_256 position 3434
[11:46:47] Possible /pseudo ‘3-keto-L-gulonate-6-phosphate decarboxylase UlaD’ at gnl|X|sample1_463 position 2219
[11:46:47] Possible /pseudo ‘Type 4 prepilin-like proteins leader peptide-processing enzyme’ at gnl|X|sample1_532 position 12547
[11:46:47] Possible /pseudo ‘Formate dehydrogenase, nitrate-inducible, major subunit’ at gnl|X|sample1_941 position 2440
[11:46:47] Possible /pseudo ‘Type-1 fimbrial protein, A chain’ at gnl|X|sample1_1089 position 955
[11:46:47] Possible /pseudo ‘HTH-type transcriptional regulator PgrR’ at gnl|X|sample1_1180 position 1666
[11:46:47] Possible /pseudo ‘Small toxic polypeptide LdrD’ at gnl|X|sample1_1641 position 921
[11:46:47] Possible /pseudo ‘Manganese transport system membrane protein MntB’ at gnl|X|sample1_1795 position 4675
[11:46:47] Possible /pseudo ‘Galactarate dehydratase (L-threo-forming)’ at gnl|X|sample1_1891 position 1152
[11:46:47] Possible /pseudo ‘Alcohol dehydrogenase 2’ at gnl|X|sample1_2550 position 704
[11:46:47] Possible /pseudo ‘putative L-ascorbate-6-phosphate lactonase UlaG’ at gnl|X|sample1_2964 position 528
[11:46:47] Found 2979 unique /gene codes.
[11:46:47] Fixed 2 duplicate /gene - argR_1 argR_2
[11:46:47] Fixed 2 duplicate /gene - mutS_1 mutS_2

(I am truncating ‘Fixing … duplicate / gene…’ output as it’s really long.
…

[11:46:47] Fixed 1048 colliding /gene names.
[11:46:47] Adding /locus_tag identifiers
[11:46:48] Assigned 7224 locus_tags to CDS and RNA features.
[11:46:48] Writing outputs to sample1/prokka/
[11:46:58] Generating annotation statistics file
[11:46:59] Generating Genbank and Sequin files
[11:46:59] Running: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y ‘Annotated using prokka 1.12 from https://github.com/tseemann/prokka’ -Z sample1/prokka/sample1.err -i sample1/prokka/sample1.fsa 2> /dev/null
[11:47:40] Could not run command: tbl2asn -V b -a r10k -l paired-ends -M n -N 1 -y ‘Annotated using prokka 1.12 from https://github.com/tseemann/prokka’ -Z sample1/prokka/sample1.err -i sample1/prokka/sample1.fsa 2> /dev/null
Makefile:134: recipe for target ‘sample1/prokka/sample1.gff’ failed
make: *** [sample1/prokka/sample1.gff] Error 2
make: *** Deleting file ‘sample1/prokka/sample1.gff’
make: Leaving directory ‘/home/ubuntu/efn/concat_reads/efn_nullarbor_run03Apr2018’
ubuntu@ebenn:~/efn$

[mattbull]

Your copy of tbl2asn has expired.

You can renew it with:

brew uninstall --ignore-dependencies tbl2asn
brew uninstall --force tbl2asn
brew install tbl2asn

Then just check that your copy of tbl2asn works by running:

tbl2asn --help

You should get the help and not an expired license warning.

[Ebenn]

Hi Matt,
Thx for your response. I have carried out the uninstallation and re-installation as above. tbl2asn is working fine now. May I resume Nullarbor? Should I re-run ‘nice make -j 1 -C /home/ubuntu/efn/concat_reads/efn_nullarbor_run03Apr2018’ now pls?

Cheers,
Ebenn

[mattbull]

Yup, carry on with nullarbor.

[Ebenn]

Thx so much again Matt. Nullarbor run has completed successfully!

Cheers,
Ebenn

[Ebenn]

Hi Matt,
Good afternoon and trust you’ve had a good weekend?
After the successful nullarbor run on four samples, I started a run with 42 samples in a screen last Friday. This morning I am noticing that it has terminated abruptly with the following error message:

ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929599-6_S24_L999_R1_001.fastq.gz’ ‘599-6/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929599-6_S24_L999_R2_001.fastq.gz’ ‘599-6/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929599-7_S49_L999_R1_001.fastq.gz’ ‘599-7/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929599-7_S49_L999_R2_001.fastq.gz’ ‘599-7/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929599-8_S3_L999_R1_001.fastq.gz’ ‘599-8/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929599-8_S3_L999_R2_001.fastq.gz’ ‘599-8/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929599-9_S67_L999_R1_001.fastq.gz’ ‘599-9/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929599-9_S67_L999_R2_001.fastq.gz’ ‘599-9/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-1_S80_L999_R1_001.fastq.gz’ ‘606-1/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-1_S80_L999_R2_001.fastq.gz’ ‘606-1/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-2_S48_L999_R1_001.fastq.gz’ ‘606-2/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-2_S48_L999_R2_001.fastq.gz’ ‘606-2/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-3_S2_L999_R1_001.fastq.gz’ ‘606-3/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-3_S2_L999_R2_001.fastq.gz’ ‘606-3/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-4_S61_L999_R1_001.fastq.gz’ ‘606-4/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-4_S61_L999_R2_001.fastq.gz’ ‘606-4/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-5_S66_L999_R1_001.fastq.gz’ ‘606-5/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-5_S66_L999_R2_001.fastq.gz’ ‘606-5/R2.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-6_S23_L999_R1_001.fastq.gz’ ‘606-6/R1.fq.gz’
ln -f -s ‘/home/ubuntu/efn/concat_reads/13-6929606-6_S23_L999_R2_001.fastq.gz’ ‘606-6/R2.fq.gz’
rm -f -r 583-P1/megahit
mkdir -p 583-P1
megahit --min-count 3 --k-list 21,31,41,53,75,97,111,127 -t 4 --memory 0.5 -1 583-P1/R1.fq.gz -2 583-P1/R2.fq.gz --out-dir 583-P1/megahit --min-contig-len 500
31.421Gb memory in total.
Using: 15.711Gb.
MEGAHIT v1.1.2
— [Sun Apr 8 14:32:40 2018] Start assembly. Number of CPU threads 4 —
— [Sun Apr 8 14:32:40 2018] Available memory: 33738153984, used: 16869076992
— [Sun Apr 8 14:32:40 2018] Converting reads to binaries —
b’ [ERROR] [sequence_manager.cpp : 151]: File(s) 583-P1/R1.fq.gz,583-P1/R2.fq.gz: Number of sequences not the same in paired files. Abort.’
Error occurs when running “megahit_asm_core buildlib”; please refer to 583-P1/megahit/log for detail
[Exit code 1]
Makefile:1395: recipe for target ‘583-P1/contigs.fa’ failed
make: *** [583-P1/contigs.fa] Error 1
make: Leaving directory ‘/home/ubuntu/efn/concat_reads/nullarbor_all_samples_run06Apr2018’
ubuntu@ebenn:~/efn/concat_reads$ nullarbor.pl --name efn_nullarbor_all_samples --mlst ecoli --ref /home/ubuntu/efn/reference_new_EcoliUMN206.fasta --input nullarbor_input_files_all_samples_06Apr2018.tab --outdir nullarbor_all_samples_run06Apr2018
Can’t locate YAML/Tiny.pm in @INC (you may need to install the YAML::Tiny module) (@INC contains: /home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/site_perl/5.22.0/x86_64-linux-thread-multi /home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/site_perl/5.22.0 /home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/5.22.0/x86_64-linux-thread-multi /home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/5.22.0 .) at /home/linuxbrew/.linuxbrew/bin/nullarbor.pl line 13.
BEGIN failed–compilation aborted at /home/linuxbrew/.linuxbrew/bin/nullarbor.pl line 13.

I wanted to check with you before attempting to install ‘YAML::Tiny module’ as the last nullarbor run completed without any hitches (after fixing the tbl2asn problem).

Standing by to hear from you please.

Grateful as always,
Ebenn

[mattbull]

You have two problems here…

---

1. megahit is failing because you have a broken pair of fastq files:

[ERROR] [sequence_manager.cpp     : 151]: File(s) 583-P1/R1.fq.gz,583-P1/R2.fq.gz: Number of sequences not the same in paired files. Abort.

Check that the paths to these files are correct in nullarbor_input_files_all_samples_06Apr2018.tab and that they are valid and correctly paired fastq files (you may have made a mistake concatenating them).

---

2. You still have a miniconda environment active and miniconda is changing @INC so that system perl modules aren’t being found:

Can’t locate YAML/Tiny.pm in @INC (you may need to install the YAML::Tiny module) (@INC contains: /home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/site_perl/5.22.0/x86_64-linux-thread-multi /home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/site_perl/5.22.0 /home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/5.22.0/x86_64-linux-thread-multi /home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/5.22.0 .) at /home/linuxbrew/.linuxbrew/bin/nullarbor.pl line 13.

Please deactivate the efn_env conda environment and to remove all traces of conda environment variables.

All of the perl modules needed for nullarbor are installed by default on GVL, so you shouldn’t need to install any perl modules, just make sure that you aren’t attached to any environments that make large-scale changes to your environment.

[Ebenn]

Dear Matt,
Many thx!
I have not activated conda environment this time, to be honest. What else might be the problem do you think?
Re the problematic reads, is it OK to remove them from the input sample list and continue without it?

Cheers,
Ebenn

[mattbull]

I have not activated conda environment this time, to be honest. What else might be the problem do you think?

To clarify - it is important that the terminal session that you used to run the nullarbor make command isn’t/wasn’t attached to a conda environment, its not important that your current terminal session doesn’t have an active conda environment.

@INC (which is where perl looks for installed modules) on a standard GVL, with no conda envs active contains:

/home/linuxbrew/.linuxbrew/Cellar/perl/5.26.1_1/lib/perl5/site_perl/5.26.1/x86_64-linux-thread-multi
/home/linuxbrew/.linuxbrew/Cellar/perl/5.26.1_1/lib/perl5/site_perl/5.26.1
/home/linuxbrew/.linuxbrew/Cellar/perl/5.26.1_1/lib/perl5/5.26.1/x86_64-linux-thread-multi
/home/linuxbrew/.linuxbrew/Cellar/perl/5.26.1_1/lib/perl5/5.26.1
/home/linuxbrew/.linuxbrew/lib/perl5/site_perl/5.26.1/x86_64-linux-thread-multi
/home/linuxbrew/.linuxbrew/lib/perl5/site_perl/5.26.1

As you can see from the logfile you pasted above, the @INC of the terminal session that you used to run the nullarbor make command contained:

/home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/site_perl/5.22.0/x86_64-linux-thread-multi
/home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/site_perl/5.22.0
/home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/5.22.0/x86_64-linux-thread-multi
/home/ubuntu/efn/miniconda3/envs/efn_env/lib/perl5/5.22.0

These are all conda paths, so unless you’re using a non-standard conda install, you may have mistakenly had an environment attached when you started the pipeline.

You can check @INC of your current terminal session by running:

perl -V

and looking at the final section of the output. If you do this before running nullarbor or the make part of the pipeline, you can be sure that you won’t run into any problems with perl modules.

---

Re the problematic reads, is it OK to remove them from the input sample list and continue without it?

It entirely depends on how important it is to you that this sample is included in the analysis. If you can forego the sample, then remove it from the tab-delimited input file. If you would like to include it, then you will need to troubleshoot and verify that the fastq files are OK before you can work on it.

[Ebenn]

Ok please.

Here is the final section of perl -V output:

DEBPKG:fixes/podman-source-date-epoch-testfix - http://bugs.debian.org/807086 Guard for building with SOURCE_DATE_EPOCH or POD_MAN_DATE set
DEBPKG:debian/devel-ppport-reproducibility - http://bugs.debian.org/801523 Sort the list of XS code files when generating RealPPPort.xs
DEBPKG:fixes/encode-unicode-bom - http://bugs.debian.org/798727 [rt.cpan.org #107043] Address https://rt.cpan.org/Public/Bug/Display.html?id=107043
DEBPKG:debian/encode-unicode-bom-doc - http://bugs.debian.org/798727 Document Debian backport of Encode::Unicode fix
DEBPKG:debian/kfreebsd-softupdates - http://bugs.debian.org/796798 Work around Debian Bug#796798
DEBPKG:fixes/autodie-scope - http://bugs.debian.org/798096 Fix a scoping issue with "no autodie" and the "system" sub
DEBPKG:debian/debugperl-compat-fix - [perl #127212] http://bugs.debian.org/810326 Disable PERL_TRACK_MEMPOOL for debugging builds
DEBPKG:fixes/CVE-2015-8607_file_spec_taint_fix - http://bugs.debian.org/810719 [perl #126862] ensure File::Spec::canonpath() preserves taint
DEBPKG:fixes/mkstemp-umask - http://bugs.debian.org/810924 [perl #127322] [e57270b] Fix umask for mkstemp(3) calls
DEBPKG:fixes/crosscompile-no-targethost - [perl #127234] Fix the Configure escape with usecrosscompile but no targethost
DEBPKG:fixes/podlators-no-encode - [rt.cpan.org #111156] Degrade gracefully if utf8 is requested but Encode is not available
DEBPKG:debian/cross-time-hires - [rt.cpan.org #111391] Add an environment variable to skip running configuration probes
DEBPKG:fixes/encode-unicode-pod - Unicode.pm: Fix POD error
DEBPKG:fixes/memoize-pod - [rt.cpan.org #89441] Fix POD errors in Memoize
DEBPKG:fixes/ok-pod - Added encoding for pod.
DEBPKG:fixes/CVE-2016-2381_duplicate_env - remove duplicate environment variables from environ
DEBPKG:fixes/CVE-2017-12837.patch - [PATCH] regcomp [perl #131582]
DEBPKG:fixes/CVE-2017-12883.patch - [PATCH] PATCH: [perl #131598]

Built under linux
Compiled at Nov 10 2017 14:39:06
@INC:
/etc/perl
/usr/local/lib/x86_64-linux-gnu/perl/5.22.1
/usr/local/share/perl/5.22.1
/usr/lib/x86_64-linux-gnu/perl5/5.22
/usr/share/perl5
/usr/lib/x86_64-linux-gnu/perl/5.22
/usr/share/perl/5.22
/usr/local/lib/site_perl
/usr/lib/x86_64-linux-gnu/perl-base

I will open a new screen and resume nullarbor from here. I’ll revert with feedback pls.

Thx
Ebenn

[Ebenn]

Hi,
Nullarbor has terminated at snippy stage with an “Error 2”. Here’s the end of the output message:

denovo.tab 580-6/denovo.tab 580-7/denovo.tab 580-8/denovo.tab 580-9/denovo.tab 583-1/denovo.tab 583-10/denovo.tab 583-11/denovo.tab 583-12/denovo.tab 583-2/denovo.tab 583-3/denovo.tab 583-4/denovo.tab 583-5/denovo.tab 583-6/denovo.tab 583-7/denovo.tab 583-8/denovo.tab 583-9/denovo.tab 599-1/denovo.tab 599-10/denovo.tab 599-11/denovo.tab 599-12/denovo.tab 599-2/denovo.tab 599-3/denovo.tab 599-4/denovo.tab 599-5/denovo.tab 599-6/denovo.tab 599-7/denovo.tab 599-8/denovo.tab 599-9/denovo.tab 606-1/denovo.tab 606-2/denovo.tab 606-3/denovo.tab 606-4/denovo.tab 606-5/denovo.tab 606-6/denovo.tab) > denovo.tab
snippy --cpus 4 --force --outdir 580-1/580-1 --ref /home/ubuntu/efn/reference_new_EcoliUMN206.fasta --R1 580-1/R1.fq.gz --R2 580-1/R2.fq.gz
[02:57:32] This is snippy 3.1
[02:57:32] Written by Torsten Seemann torsten.seemann@gmail.com
[02:57:32] Obtained from https://github.com/tseemann/snippy
[02:57:32] Detected operating system: linux
[02:57:32] Enabling bundled linux tools.
[02:57:32] Found bwa - /home/linuxbrew/.linuxbrew/bin/bwa
[02:57:32] Found samtools - /home/linuxbrew/.linuxbrew/bin/samtools
[02:57:32] Found tabix - /home/linuxbrew/.linuxbrew/bin/tabix
[02:57:32] Found bgzip - /home/linuxbrew/.linuxbrew/bin/bgzip
[02:57:32] Found snpEff - /home/linuxbrew/.linuxbrew/Cellar/snippy/3.1/bin/…/binaries/noarch/snpEff
[02:57:32] Found java - /home/linuxbrew/.linuxbrew/bin/java
[02:57:32] Found gzip - /bin/gzip
[02:57:32] Found parallel - /home/linuxbrew/.linuxbrew/bin/parallel
[02:57:32] Found freebayes - /home/linuxbrew/.linuxbrew/bin/freebayes
[02:57:32] Found freebayes-parallel - /home/linuxbrew/.linuxbrew/bin/freebayes-parallel
[02:57:32] Found fasta_generate_regions.py - /home/linuxbrew/.linuxbrew/bin/fasta_generate_regions.py
[02:57:32] Found vcfstreamsort - /home/linuxbrew/.linuxbrew/bin/vcfstreamsort
[02:57:32] Found vcfuniq - /home/linuxbrew/.linuxbrew/bin/vcfuniq
[02:57:32] Found vcffirstheader - /home/linuxbrew/.linuxbrew/bin/vcffirstheader
[02:57:32] Found vcf-consensus - /home/linuxbrew/.linuxbrew/bin/vcf-consensus
[02:57:32] Found snippy-vcf_to_tab - /home/linuxbrew/.linuxbrew/Cellar/snippy/3.1/bin/snippy-vcf_to_tab
[02:57:32] Found snippy-vcf_report - /home/linuxbrew/.linuxbrew/Cellar/snippy/3.1/bin/snippy-vcf_report
[02:57:32] Found snippy-vcf_filter - /home/linuxbrew/.linuxbrew/Cellar/snippy/3.1/bin/snippy-vcf_filter
[02:57:32] Using reference: /home/ubuntu/efn/reference_new_EcoliUMN206.fasta
[02:57:32] Treating reference as ‘fasta’ format.
[02:57:32] Will use 4 CPU cores.
[02:57:32] Using read file: /home/ubuntu/efn/concat_reads/nullarbor_run08Apr2018/580-1/R1.fq.gz
[02:57:32] Using read file: /home/ubuntu/efn/concat_reads/nullarbor_run08Apr2018/580-1/R2.fq.gz
[02:57:32] Creating folder: 580-1/580-1
[02:57:32] Changing working directory: 580-1/580-1
[02:57:32] Creating reference folder: reference
[02:57:32] Extracting FASTA and GFF from reference.
[02:57:33] Wrote 1 sequences to ref.fa
[02:57:33] Wrote 0 features to ref.gff
[02:57:33] Freebayes will process 16 chunks of 330554 bp, 4 chunks at a time.
[02:57:33] Using BAM RG (Read Group) ID: snps
[02:57:33] Running: samtools faidx reference/ref.fa 2>> snps.log
[02:57:33] Running: bwa index reference/ref.fa 2>> snps.log
[02:57:37] Running: mkdir reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa 2>> snps.log
[02:57:37] Running: mkdir reference/ref && bgzip -c reference/ref.gff > reference/ref/genes.gff.gz 2>> snps.log
[02:57:37] Running: (bwa mem -v 2 -M -R ‘@RG ID:snps SM:snps’ -t 4 reference/ref.fa /home/ubuntu/efn/concat_reads/nullarbor_run08Apr2018/580-1/R1.fq.gz /home/ubuntu/efn/concat_reads/nullarbor_run08Apr2018/580-1/R2.fq.gz | samtools view -@ 4 -F 3844 -q 60 -S -b -u -T reference/ref.fa - | samtools sort -O bam -o snps.bam -@ 4 -) 2>> snps.log
[02:57:37] Running: samtools index snps.bam 2>> snps.log
[02:57:37] Running: samtools depth -q 20 snps.bam | bgzip > snps.depth.gz 2>> snps.log
[02:57:37] Running: tabix -s 1 -b 2 -e 2 snps.depth.gz 2>> snps.log
[02:57:37] Running: fasta_generate_regions.py reference/ref.fa.fai 330554 > reference/ref.txt 2>> snps.log
[02:57:37] Error running command, check 580-1/580-1/snps.log
Makefile:33: recipe for target ‘580-1/580-1/snps.tab’ failed
make: *** [580-1/580-1/snps.tab] Error 2
make: Leaving directory ‘/home/ubuntu/efn/concat_reads/nullarbor_run08Apr2018’
ubuntu@ebenn:~/efn/concat_reads$

Your kind assistance is greatly appreciated as always

[Ebenn]

Error running command, check 580-1/580-1/snps.log

Here’s the content of 580-1/580-1/snps.log please:

ubuntu@ebenn:~/efn/concat_reads/nullarbor_run08Apr2018/580-1/580-1$ cat snps.log

samtools faidx reference/ref.fa

bwa index reference/ref.fa

[bwa_index] Pack FASTA… 0.07 sec
[bwa_index] Construct BWT for the packed sequence…
[bwa_index] 2.52 seconds elapse.
[bwa_index] Update BWT… 0.04 sec
[bwa_index] Pack forward-only FASTA… 0.03 sec
[bwa_index] Construct SA from BWT and Occ… 0.96 sec
[main] Version: 0.7.17-r1188
[main] CMD: bwa index reference/ref.fa
[main] Real time: 3.886 sec; CPU: 3.628 sec

mkdir reference/genomes && cp -f reference/ref.fa reference/genomes/ref.fa

mkdir reference/ref && bgzip -c reference/ref.gff > reference/ref/genes.gff.gz

(bwa mem -v 2 -M -R ‘@RG ID:snps SM:snps’ -t 4 reference/ref.fa /home/ubuntu/efn/concat_reads/nullarbor_run08Apr2018/580-1/R1.fq.gz /home/ubuntu/efn/concat_reads/nullarbor_run08Apr2018/580-1/R2.fq.gz | samtools view -@ 4 -F 3844 -q 60 -S -b -u -T reference/ref.fa - | samtools sort -O bam -o snps.bam -@ 4 -)

[E::bwa_set_rg] the read group line contained literal characters – replace with escaped tabs: \t

samtools index snps.bam

samtools depth -q 20 snps.bam | bgzip > snps.depth.gz

tabix -s 1 -b 2 -e 2 snps.depth.gz

fasta_generate_regions.py reference/ref.fa.fai 330554 > reference/ref.txt

File “/home/linuxbrew/.linuxbrew/bin/fasta_generate_regions.py”, line 7
print "usage: ", sys.argv[0], " "
^
SyntaxError: Missing parentheses in call to ‘print’. Did you mean print(int "usage: ", sys.argv[0], " ")?

(end of snp.log content)

Thx
Ebenn

[mattbull]

The way that print function is called changed between Python 2 and 3. The error below is caused by trying to run a program written in Python 2 (fasta_generate_regions.py) using the Python 3 interpreter.

SyntaxError: Missing parentheses in call to ‘print’. Did you mean print(int "usage: ", sys.argv[0], "  ")?

I suspect that conda is still interfering with your environment. The default miniconda3 python interpreter is currently something like version 3.6.4, and the default Ubuntu 16.04 interpreter is something like version 2.7.12.

If you used the automatic miniconda installer, it gives you the option to prepend the miniconda bin directory to the PATH by writing the following to the end ~/.bashrc:

# added by Miniconda3 installer
export PATH="/home/ubuntu/miniconda3/bin:$PATH"

I suspect that you will need to comment out this line then logout and log back in to your VM for the changes to take effect to fix this problem.

---

You can check python interpreter your session is using with:

which python
python --version

For nullarbor to run smoothly, you want a Python version of 2.x.x.

---

I’d suggest that if you want to use miniconda, it need to be the sole package manager installed, or at least only using software from inside the environment, not system and linuxbrew packages.

I suspect that you’ll get on better using miniconda on a clean Ubuntu 16.04 install, rather than GVL.

[Ebenn]

export PATH="/home/ubuntu/miniconda3/bin:$PATH"

Hi Matt,
You’re right, my python version is 3.6.4. I changed directory to /home/ubuntu/efn/miniconda3/bin/.bashrc and added 'export PATH="/home/ubuntu/miniconda3/bin:$PATH", rebooted my VM and tried to resume nullarbor, but the error persists.

Please can you guide me through a “clean ubunt 16.04 install” ? I have no idea how to proceed.

Thx,
Ebenn

[mattbull]

The problem is with the file:

/home/ubuntu/.bashrc

It has a line at the bottom which reads:

export PATH="/home/ubuntu/miniconda3/bin:$PATH"

Please use nano, to edit this file with the following command:

nano /home/ubuntu/.bashrc

and replace the line that reads:

export PATH="/home/ubuntu/miniconda3/bin:$PATH"

with:

#export PATH="/home/ubuntu/miniconda3/bin:$PATH"

When you have done that, and pressed Ctrl+X and followed the prompts to save the file and exit nano, disconnect and reconnect to your VM.

[Ebenn]

and replace the line that reads:

export PATH="/home/ubuntu/miniconda3/bin:$PATH"

with:

#export PATH="/home/ubuntu/miniconda3/bin:$PATH"

When you have done that, and pressed Ctrl+X and followed the prompts to save the file and exit nano, disconnect and reconnect to your VM.

Thx Matt,
This is done, and nullarbor resumed again. But I’m afraid there is an error about creating the core.aln. Here is what it says:

–aformat=s Output alignment format: nexus fasta phylip maf clustalw … (default ‘fasta’).
Makefile:2302: recipe for target ‘core.aln’ failed
make: *** [core.aln] Error 1
make: Leaving directory ‘/home/ubuntu/efn/concat_reads/nullarbor_run08Apr2018’
ubuntu@ebenn:~/efn/concat_reads$ ls

Any idea what might this be please?

Thx,
Ebenn

[mattbull]

I suspect that the problem is this:

[E::bwa_set_rg] the read group line contained literal characters – replace with escaped tabs: \t

But its difficult to say exactly because this run has stopped and started so many times. I suggest that you install the latest version of snippy (v3.2):

brew upgrade snippy

Then completely remove the output directory from your nullarbor run and start it again fresh.

[Ebenn]

brew upgrade snippy

Thx Matt!

I have upgraded snippy, but it gave several warnings! Please see below:

Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/nullarbor.rb:16:in `class:Nullarbor’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/nullarbor.rb:17:in `class:Nullarbor’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/nullarbor.rb:18:in `class:Nullarbor’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/nullarbor.rb:19:in `class:Nullarbor’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/abricate.rb:14:in `class:Abricate’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/abricate.rb:15:in `class:Abricate’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/abricate.rb:16:in `class:Abricate’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/abricate.rb:17:in `class:Abricate’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/snippy.rb:12:in `class:Snippy’
Please report this to the tseemann/bioinformatics-linux tap!

Warning: Calling ‘depends_on … => :perl’ is deprecated!
There is no replacement.
/home/linuxbrew/.linuxbrew/Library/Taps/tseemann/homebrew-bioinformatics-linux/Formula/snippy.rb:13:in `class:Snippy’
Please report this to the tseemann/bioinformatics-linux tap!

==> Upgrading 1 outdated package, with result:
tseemann/bioinformatics-linux/snippy 3.1 -> 3.2
Error: No available formula with the name “vcflib” (dependency of tseemann/bioinformatics-linux/snippy)

Not sure if it’s ok to proceed?

Thx,
Ebenn

[mattbull]

The warnings aren’t a problem, but you’ll need these two taps before you can upgrade snippy:

brew tap brewsci/science
brew tap brewsci/bio

Then you can run:

brew upgrade snippy

Ignoring the warnings.