I cannot use mitobim to analyse my data

311 views

Skip to first unread message

pass...@ku.th

unread,

Oct 25, 2017, 5:00:47 PM10/25/17

to MITObim-users

Dear Christoph,

I am trying to use mitobim to assemble my mitochondrial genome sequences obtained from Illumina sequencer. I first followed your instruction to set up mitobim docker on my google cloud (1 vCPU, 3.75 GB memory, 10 GB standard persistent boot and local disk). The setting process went just fine and I could smoothly run your test data. However, when I applied the same process in tutorial 1 step 1 on my data, I got the following error message and obtained no assembled sequence. What should I do to fix the problem?

Please cite: Chevreux, B., Wetter, T. and Suhai, S. (1999), Genome Sequence

Assembly Using Trace Signals and Additional Sequence Information.

Computer Science and Biology: Proceedings of the German Conference on

Bioinformatics (GCB) 99, pp. 45-56.

To (un-)subscribe the MIRA mailing lists, see:

http://www.chevreux.org/mira_mailinglists.html

After subscribing, mail general questions to the MIRA talk mailing list:

mira...@freelists.org

To report bugs or ask for features, please use the SourceForge ticketing

system at:

http://sourceforge.net/p/mira-assembler/tickets/

This ensures that requests do not get lost.

Compiled by: bach

Fri Apr 18 14:57:20 CEST 2014

On: Linux vk10464 2.6.32-41-generic #94-Ubuntu SMP Fri Jul 6 18:00:34 UTC 2012 x86_64 GNU/Linux

Compiled in boundtracking mode.

Compiled in bugtracking mode.

Compiled with ENABLE64 activated.

Runtime settings (sorry, for debug):

Size of size_t : 8

Size of uint32 : 4

Size of uint32_t: 4

Size of uint64 : 8

Size of uint64_t: 8

Current system: Linux 901418c34dee 4.10.0-37-generic #41~16.04.1-Ubuntu SMP Fri Oct 6 22:42:59 UTC 2017 x86_64 x86_64 x86_64 GNU/Linux

Looking for files named in data ...Pushing back filename: "reference.fa"

Pushing back filename: "reads.fastq"

Manifest:

projectname: initial-mapping-testpool-to-Lsubstriata-mt

job: genome,mapping,accurate

parameters: -NW:mrnl=0 -AS:nop=1 SOLEXA_SETTINGS -CO:msr=no

Manifest load entries: 2

MLE 1:

RGID: 1

RGN: SN: Lsubstriata-mt-genome

SP: SPio: 0 SPC: 0 IF: -1 IT: -1 TSio: 0

ST: 5 (Text) namschem: 6 SID: 0

DQ: 30

BB: 1 Rail: 0 CER: 0

reference.fa MLE 2:

RGID: 2

RGN: reads SN: testpool

SP: SPio: 0 SPC: 0 IF: -1 IT: -1 TSio: 0

ST: 6 (Solexa) namschem: 4 SID: 0

DQ: 30

BB: 0 Rail: 0 CER: 0

reads.fastq

Parameters parsed without error, perfect.

-CL:pec and -CO:emeas1clpec are set, setting -CO:emea values to 1.

------------------------------------------------------------------------------

Parameter settings seen for:

Sanger data

Used parameter settings:

General (-GE):

Project name : initial-mapping-testpool-to-Lsubstriata-mt

Number of threads (not) : 2

Automatic memory management (amm) : yes

Keep percent memory free (kpmf) : 15

Max. process size (mps) : 0

EST SNP pipeline step (esps) : 0

Colour reads by hash frequency (crhf) : yes

Load reads options (-LR):

Wants quality file (wqf) : [sxa] yes

Filecheck only (fo) : no

Assembly options (-AS):

Number of passes (nop) : 1

Skim each pass (sep) : yes

Maximum number of RMB break loops (rbl) : 1

Maximum contigs per pass (mcpp) : 0

Minimum read length (mrl) : [sxa] 20

Minimum reads per contig (mrpc) : [sxa] 10

Enforce presence of qualities (epoq) : [sxa] yes

Automatic repeat detection (ard) : yes

Coverage threshold (ardct) : [sxa] 2

Minimum length (ardml) : [sxa] 200

Grace length (ardgl) : [sxa] 20

Use uniform read distribution (urd) : no

Start in pass (urdsip) : 3

Cutoff multiplier (urdcm) : [sxa] 1.5

Spoiler detection (sd) : yes

Last pass only (sdlpo) : yes

Use genomic pathfinder (ugpf) : yes

Use emergency search stop (uess) : yes

ESS partner depth (esspd) : 500

Use emergency blacklist (uebl) : yes

Use max. contig build time (umcbt) : no

Build time in seconds (bts) : 10000

Strain and backbone options (-SB):

Bootstrap new backbone (bnb) : yes

Start backbone usage in pass (sbuip) : 0

Backbone rail from strain (brfs) :

Backbone rail length (brl) : 0

Backbone rail overlap (bro) : 0

Trim overhanging reads (tor) : yes

(Also build new contigs (abnc)) : no

Dataprocessing options (-DP):

Use read extensions (ure) : [sxa] no

Read extension window length (rewl) : [sxa] 30

Read extension w. maxerrors (rewme) : [sxa] 2

First extension in pass (feip) : [sxa] 0

Last extension in pass (leip) : [sxa] 0

Clipping options (-CL):

SSAHA2 or SMALT clipping:

Gap size (msvsgs) : [sxa] 1

Max front gap (msvsmfg) : [sxa] 2

Max end gap (msvsmeg) : [sxa] 2

Strict front clip (msvssfc) : [sxa] 0

Strict end clip (msvssec) : [sxa] 0

Possible vector leftover clip (pvlc) : [sxa] no

maximum len allowed (pvcmla) : [sxa] 18

Min qual. threshold for entire read (mqtfer): [sxa] 0

Number of bases (mqtfernob) : [sxa] 15

Quality clip (qc) : [sxa] no

Minimum quality (qcmq) : [sxa] 20

Window length (qcwl) : [sxa] 30

Bad stretch quality clip (bsqc) : [sxa] no

Minimum quality (bsqcmq) : [sxa] 5

Window length (bsqcwl) : [sxa] 20

Masked bases clip (mbc) : [sxa] no

Gap size (mbcgs) : [sxa] 5

Max front gap (mbcmfg) : [sxa] 12

Max end gap (mbcmeg) : [sxa] 12

Lower case clip front (lccf) : [sxa] no

Lower case clip back (lccb) : [sxa] no

Clip poly A/T at ends (cpat) : [sxa] no

Keep poly-a signal (cpkps) : [sxa] no

Minimum signal length (cpmsl) : [sxa] 12

Max errors allowed (cpmea) : [sxa] 1

Max gap from ends (cpmgfe) : [sxa] 9

Clip 3 prime polybase (c3pp) : [sxa] yes

Minimum signal length (c3ppmsl) : [sxa] 15

Max errors allowed (c3ppmea) : [sxa] 3

Max gap from ends (c3ppmgfe) : [sxa] 9

Clip known adaptors right (ckar) : [sxa] yes

Ensure minimum left clip (emlc) : [sxa] no

Minimum left clip req. (mlcr) : [sxa] 0

Set minimum left clip to (smlc) : [sxa] 0

Ensure minimum right clip (emrc) : [sxa] no

Minimum right clip req. (mrcr) : [sxa] 10

Set minimum right clip to (smrc) : [sxa] 20

Apply SKIM chimera detection clip (ascdc) : no

Apply SKIM junk detection clip (asjdc) : no

Propose end clips (pec) : [sxa] yes

Bases per hash (pecbph) : 31

Handle Solexa GGCxG problem (pechsgp) : yes

Front freq (pffreq) : [sxa] 0

Back freq (pbfreq) : [sxa] 0

Minimum kmer for forward-rev (pmkfr) : 1

Front forward-rev (pffore) : [sxa] yes

Back forward-rev (pbfore) : [sxa] yes

Front conf. multi-seq type (pfcmst) : [sxa] yes

Back conf. multi-seq type (pbcmst) : [sxa] yes

Front seen at low pos (pfsalp) : [sxa] no

Back seen at low pos (pbsalp) : [sxa] no

Clip bad solexa ends (cbse) : [sxa] yes

Search PhiX174 (spx174) : [sxa] yes

Filter PhiX174 (fpx174) : [sxa] no

Rare kmer mask (rkm) : [sxa] 0

Parameters for SKIM algorithm (-SK):

Number of threads (not) : 2

Also compute reverse complements (acrc) : yes

Bases per hash (bph) : 10

Automatic increase per pass (bphaipp) : 1

Automatic incr. cov. threshold (bphaict): 20

Hash save stepping (hss) : 1

Percent required (pr) : [sxa] 60

Max hits per read (mhpr) : 2000

Max megahub ratio (mmhr) : 0

SW check on backbones (swcob) : yes

Max hashes in memory (mhim) : 15000000

MemCap: hit reduction (mchr) : 4096

Parameters for Hash Statistics (-HS):

Freq. cov. estim. min (fcem) : 0

Freq. estim. min normal (fenn) : 0.4

Freq. estim. max normal (fexn) : 1.6

Freq. estim. repeat (fer) : 1.9

Freq. estim. heavy repeat (fehr) : 8

Freq. estim. crazy (fecr) : 20

Mask nasty repeats (mnr) : no

Nasty repeat ratio (nrr) : 100

Nasty repeat coverage (nrc) : 0

Lossless digital normalisation (ldn) : no

Repeat level in info file (rliif) : 6

Million hashes per buffer (mhpb) : 16

Rare kmer early kill (rkek) : no

Pathfinder options (-PF):

Use quick rule (uqr) : [sxa] yes

Quick rule min len 1 (qrml1) : [sxa] -90

Quick rule min sim 1 (qrms1) : [sxa] 100

Quick rule min len 2 (qrml2) : [sxa] -80

Quick rule min sim 2 (qrms2) : [sxa] 100

Backbone quick overlap min len (bqoml) : [sxa] 20

Max. start cache fill time (mscft) : 5

Align parameters for Smith-Waterman align (-AL):

Bandwidth in percent (bip) : [sxa] 20

Bandwidth max (bmax) : [sxa] 80

Bandwidth min (bmin) : [sxa] 20

Minimum score (ms) : [sxa] 15

Minimum overlap (mo) : [sxa] 20

Minimum relative score in % (mrs) : [sxa] 60

Solexa_hack_max_errors (shme) : [sxa] -1

Extra gap penalty (egp) : [sxa] no

extra gap penalty level (egpl) : [sxa] reject_codongaps

Max. egp in percent (megpp) : [sxa] 100

Contig parameters (-CO):

Name prefix (np) : initial-mapping-testpool-to-Lsubstriata-mt

Reject on drop in relative alignment score in % (rodirs) : [sxa] 30

Mark repeats (mr) : yes

Only in result (mroir) : no

Assume SNP instead of repeats (asir) : no

Minimum reads per group needed for tagging (mrpg) : [sxa] 3

Minimum neighbour quality needed for tagging (mnq) : [sxa] 20

Minimum Group Quality needed for RMB Tagging (mgqrt) : [sxa] 30

End-read Marking Exclusion Area in bases (emea) : [sxa] 1

Set to 1 on clipping PEC (emeas1clpec) : yes

Also mark gap bases (amgb) : [sxa] yes

Also mark gap bases - even multicolumn (amgbemc) : [sxa] yes

Also mark gap bases - need both strands (amgbnbs): [sxa] yes

Force non-IUPAC consensus per sequencing type (fnicpst) : [sxa] no

Merge short reads (msr) : [sxa] no

Max errors (msrme) : [sxa] 0

Keep ends unmerged (msrkeu) : [sxa] -1

Gap override ratio (gor) : [sxa] 66

Edit options (-ED):

Mira automatic contig editing (mace) : yes

Edit kmer singlets (eks) : yes

Edit homopolymer overcalls (ehpo) : [sxa] no

Misc (-MI):

Large contig size (lcs) : 500

Large contig size for stats (lcs4s) : 5000

I know what I do (ikwid) : no

Extra flag 1 / sanity track check (ef1) : no

Extra flag 2 / dnredreadsatpeaks (ef2) : yes

Extra flag 3 / pelibdisassemble (ef3) : yes

Extended log (el) : no

Nag and Warn (-NW):

Check NFS (cnfs) : stop

Check multi pass mapping (cmpm) : stop

Check template problems (ctp) : stop

Check duplicate read names (cdrn) : stop

Check max read name length (cmrnl) : stop

Max read name length (mrnl) : 0

Check average coverage (cac) : stop

Average coverage value (acv) : 160

Directories (-DI):

Top directory for writing files : initial-mapping-testpool-to-Lsubstriata-mt_assembly

For writing result files : initial-mapping-testpool-to-Lsubstriata-mt_assembly/initial-mapping-testpool-to-Lsubstriata-mt_d_results

For writing result info files : initial-mapping-testpool-to-Lsubstriata-mt_assembly/initial-mapping-testpool-to-Lsubstriata-mt_d_info

For writing tmp files : initial-mapping-testpool-to-Lsubstriata-mt_assembly/initial-mapping-testpool-to-Lsubstriata-mt_d_tmp

Tmp redirected to (trt) :

For writing checkpoint files : initial-mapping-testpool-to-Lsubstriata-mt_assembly/initial-mapping-testpool-to-Lsubstriata-mt_d_chkpt

Output files (-OUTPUT/-OUT):

Save simple singlets in project (sssip) : [sxa] no

Save tagged singlets in project (stsip) : [sxa] yes

Remove rollover tmps (rrot) : yes

Remove tmp directory (rtd) : no

Result files:

Saved as CAF (orc) : yes

Saved as MAF (orm) : yes

Saved as FASTA (orf) : yes

Saved as GAP4 (directed assembly) (org) : no

Saved as phrap ACE (ora) : no

Saved as GFF3 (org3) : no

Saved as HTML (orh) : no

Saved as Transposed Contig Summary (ors) : yes

Saved as simple text format (ort) : no

Saved as wiggle (orw) : yes

Temporary result files:

Saved as CAF (otc) : yes

Saved as MAF (otm) : no

Saved as FASTA (otf) : no

Saved as GAP4 (directed assembly) (otg) : no

Saved as phrap ACE (ota) : no

Saved as HTML (oth) : no

Saved as Transposed Contig Summary (ots) : no

Saved as simple text format (ott) : no

Extended temporary result files:

Saved as CAF (oetc) : no

Saved as FASTA (oetf) : no

Saved as GAP4 (directed assembly) (oetg) : no

Saved as phrap ACE (oeta) : no

Saved as HTML (oeth) : no

Save also singlets (oetas) : no

Alignment output customisation:

TEXT characters per line (tcpl) : 60

HTML characters per line (hcpl) : 60

TEXT end gap fill character (tegfc) :

HTML end gap fill character (hegfc) :

File / directory output names:

CAF : initial-mapping-testpool-to-Lsubstriata-mt_out.caf

MAF : initial-mapping-testpool-to-Lsubstriata-mt_out.maf

FASTA : initial-mapping-testpool-to-Lsubstriata-mt_out.unpadded.fasta

FASTA quality : initial-mapping-testpool-to-Lsubstriata-mt_out.unpadded.fasta.qual

FASTA (padded) : initial-mapping-testpool-to-Lsubstriata-mt_out.padded.fasta

FASTA qual.(pad): initial-mapping-testpool-to-Lsubstriata-mt_out.padded.fasta.qual

GAP4 (directory): initial-mapping-testpool-to-Lsubstriata-mt_out.gap4da

ACE : initial-mapping-testpool-to-Lsubstriata-mt_out.ace

HTML : initial-mapping-testpool-to-Lsubstriata-mt_out.html

Simple text : initial-mapping-testpool-to-Lsubstriata-mt_out.txt

TCS overview : initial-mapping-testpool-to-Lsubstriata-mt_out.tcs

Wiggle : initial-mapping-testpool-to-Lsubstriata-mt_out.wig

------------------------------------------------------------------------------

Deleting old directory initial-mapping-testpool-to-Lsubstriata-mt_assembly ... done.

Creating directory initial-mapping-testpool-to-Lsubstriata-mt_assembly ... done.

Creating directory initial-mapping-testpool-to-Lsubstriata-mt_assembly/initial-mapping-testpool-to-Lsubstriata-mt_d_results ... done.

Creating directory initial-mapping-testpool-to-Lsubstriata-mt_assembly/initial-mapping-testpool-to-Lsubstriata-mt_d_info ... done.

Creating directory initial-mapping-testpool-to-Lsubstriata-mt_assembly/initial-mapping-testpool-to-Lsubstriata-mt_d_chkpt ... done.

Creating directory initial-mapping-testpool-to-Lsubstriata-mt_assembly/initial-mapping-testpool-to-Lsubstriata-mt_d_tmp ... done.

Tmp directory is not on a NFS mount, good.