[mira_talk] Re: mira_talk Digest V2 #23

Thanks for the reply, I am running the assembly on another server thus springs 
another problem. But, for completeness this is what I found involving the first 
question. 

1---
> 1) Could you please send me the FASTA file you are loading the backbone from? 
> There's something strange in the log as MIRA says it loaded two sequences but 
> just added rails for one backbone. This is not normal.

I discovered a mistake in the file. I assume Mira is regonizing the “>” as the 
fasta header. When I merged a genome and plasmid file (> merge file1..) to 
create backbone and did not remove the bottom tag Ex, fig 1.  
>gi|150392480|ref|NC_009632.1| Staphylococcus aureus subsp. aureus JH1, 
>complete genome

AATTT .....
. >>>>>>NC_009619.fna
end of file. 

2----
> 2) If you are running a 64 bit executable: what's the system memory and how 
> many reads are in the Solexa file?

Yes. I did not have enough memory for the solexa reads (14, 771, 764 
reads)(ex2), and have switched to a second server with 16 gigs thus my second 
issue arose. 
Cmd:> free 
Running in 64 bit mode.
 ... Dump from /proc/meminfo
--------------------------------------------------------------------------------
MemTotal:      2059816 kB , MemFree:        415604 kB
 ,  Buffers:         37200 kB, Cached:        1142612 kB
 , SwapCached:      14700 kB


3---

Input files:
sa2_in.solexa.fasta
sa2_backbone_in.gbf
sa2_straindata_in.txt

command:
> mira --project=sa2 --job=mapping,genome,normal,solexa 
> -SB:lsd=yes:bsn=sa2:bft=gbf:bbq=30 >10_log_assembly_sa.txt


For nearly each read, it repeats the following for example.
: Solexa: Filter out (A hard) sa2|218
: Solexa: Filter out (A hard) sa2|819
: Solexa: Filter out (A hard) sa2|894
.... then the following 
: Short length: sa2|14771243 (sxa): only 0 good bases, need: 20. No paired end 
par
: tner, rejected.
: Short length: sa2|14771244 (sxa): only 0 good bases, need: 20. No paired end 
par
: tner, rejected.

until moving to phase 1 were there system froze. 

This is were the system freezes. I am unsure if it is a memory issue or 
hardware issue. 
Currently running on a...

Intel(R)Xeon(R) CPU   X5355
2550.00 Mhz
4096 KB 
16 gb ram
Ubunto 8.04 Hardy.




Thanks for any help in advance

Alex Washington
Graduate Student:VCU
alexwashington@xxxxxxxxx



> -----Original Message-----
> From: ecartis@xxxxxxxxxxxxx
> Sent: Sat, 14 Mar 2009 01:10:31 -0400 (EDT)
> To: ecartis@xxxxxxxxxxxxx
> Subject: mira_talk Digest V2 #23
> 
> mira_talk Digest      Fri, 13 Mar 2009        Volume: 02  Issue: 023
> 
> In This Issue:
>               [mira_talk] Mira Question: Solexa reads...memory error or bu
>               [mira_talk] Re: Mira Question: Solexa reads...memory error o
> 
> ----------------------------------------------------------------------
> 
> Date: Fri, 13 Mar 2009 11:55:27 -0800
> From: Alex Washington <alexwashington@xxxxxxxxx>
> Subject: [mira_talk] Mira Question: Solexa reads...memory error or bug?
> 
> Mira community,
> Greetings, I have throughly searched through the Mira package for an
> answer to this problem but to no avail.
> 
> After Running Mira with the following command:
> 
>    input files:
>      sa2_in.solexa.fasta
> 
>      sa2_straindata_in.txt
> 
>      sa2_backbone_in.fasta
>        ***no qualilty files available in project, thus using default!
> 
>    command:
>     > mira --project=sa2 --job=mapping,genome,normal,solexa
> -SB:lsd=yes:bsn=sa2:bftzsta:bbq0 >2_log_assembly_sa.txt
> 
> 
> 
> the program quits and with the following error....
> 
>     b?? error ... out of memory...if you have any questions please send
> the   last 1000 lines of output log to the author, b??
> 
> 
> I should have sufficient amount of memory thus this error is unexpected.
> Is this a bug?
> 
> 
> Project Description:
> S. aureus
> Illumina Solexa data
> No. of reads:  14, 771, 764 reads
> 
> 
> Attached: alex_log_assembly.txt
> 
> 
> Thanks
> 
> Alex Washintong
> Graduate Student:VCU
> alexwashington@xxxxxxxxx
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> ------------------------------
> 
> From: Bastien Chevreux <bach@xxxxxxxxxxxx>
> Subject: [mira_talk] Re: Mira Question: Solexa reads...memory error or
> bug?
> Date: Fri, 13 Mar 2009 23:09:05 +0100
> 
> On Friday 13 March 2009 Alex Washington wrote:
>> Greetings, I have throughly searched through the Mira package for an
>> answer
>> to this problem but to no avail.
>> [...]
> 
> Hello Alex,
> 
> the manuals for Solexa could be ... somewhat more extensive, I agree :-)
> 
> However, your problem seems to be something peculiar. Let's start with
> the
> side note (which are probably not interesting to you but might be
> important to
> me):
> 
> 1) Could you please send me the FASTA file you are loading the backbone
> from?
> There's something strange in the log as MIRA says it loaded two sequences
> but
> just added rails for one backbone. This is not normal.
> 2) It looks like your backbone is the sequence from a GenBank entry. For
> prokaryotic entries, MIRA has no problem loading a GenBank entry
> directly,
> including the complete annotations ... which opens up a some neat goodies
> further down the analysis pipeline. You might want to try that some day.
> 
> Now to your out of memory problem: it comes at a very surprising point,
> just
> when MIRA allocates space to get the sequences loaded.
> 
> Questions:
> 1) Would you be running a 32 bit executable? Then, even if your system
> has
> dozens of gigabytes, a process can use only 2.7 to 3.5 GiB. Which might
> lead
> to out of memory.
> 2) If you are running a 64 bit executable: what's the system memory and
> how
> many reads are in the Solexa file?
> 
> Now, before answering that yourself: could you please download
> 
>   http://www.chevreux.org/tmp/mira_2.9.42x1_dev_linux-gnu_x86_64.tar.bz2
> 
> and send me the output of that version? I've added the needed things to
> the
> log so I can look it up myself.
> 
> Besides, in 2.9.42 I've activated new Solexa clipping routines which are
> way
> faster and better than the old ones. With this version I assembled and
> performed mutation analyses on 5 Solexa projects on Wednesday and
> Thursday, so
> it's quite well tested.
> 
> Regards,
>   Bastien
> 
> 
> ------------------------------
> 
> End of mira_talk Digest V2 #23
> ******************************

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Short length: sa2|14771164 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771177 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771179 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771193 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771196 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771204 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771212 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771223 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771232 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771240 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771243 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.
Short length: sa2|14771244 (sxa): only 0 good bases, need: 20. No paired end 
partner, rejected.

===========================================================================
Pool statistics:
Backbones: 1    Backbone rails: 48440

                Sanger  454     Solexa  SOLiD
                ---------------------------------
Total reads     0       0       14771262        0
Reads wo qual   0       0       0       0
Used reads      0       0       14414891        0
Avg len used    0       0       41      0

With strain     0       0       0       0
W/o clips       0       0       14771262        0
===========================================================================

Localtime: Mon Mar 16 14:01:29 2009

Seeing strain 1: "sa2"
Generated 1 unique strain ids for 14819703 reads.
Strain "default" has 14771262 reads.
Strain "sa2" has 1 reads.

Writing temporary hstat files:
 [0%] ....|.... [10%] ....|.... [20%] ....|.... [30%] ....|.... [40%] ....|.... 
[50%] ....|.... [60%] ....|.... [70%] ....|.... [80%] ....|.... [90%] ....|.... 
[100%] done

Analysing hstat files:
 [0%] ....|.... [10%] ....|.... [20%] ....|.... [30%] ....|.... [40%] ....|.... 
[50%] ....|.... [60%] ....|.... [70%] ....|.... [80%] ....|.... [90%] ....|.... 
[100%] 
Assigning values:
Localtime: Mon Mar 16 14:11:25 2009
 [0%] ....|.... [10%] ....|.... [20%] ....|.... [30%] ....|.... [40%] ....|.... 
[50%] ....|.... [60%] ....|.... [70%] ....|.... [80%] ....|.... [90%] ....|.... 
[100%] done
Localtime: Mon Mar 16 14:15:14 2009
Localtime: Mon Mar 16 14:15:15 2009

Looking for proposed cutbacks ... done.
Performed clips:
        Num reads cliped left: 4387582
        Num reads cliped right: 6179180
        Num reads completely killed: 1456590

Localtime: Mon Mar 16 14:15:27 2009


Searching for possible overlaps (only against backbone):
Localtime: Mon Mar 16 14:15:27 2009
Now running threaded and partitioned skimmer with 34 partitions in 2 threads:
 [0%] ....|.... [10%] ....|.... [20%] ....|.... [30%] ....|.... [40%] ....|.... 
[50%] ....|.... [60%] ....|.... [70%] ....|.... [80%] ....|.... [90%] ....|.... 
[100%]  done.

Skim summary:
        accepted: 56399943
        possible: 605354678
        permbans: 0

Hits chosen: 50442493

Localtime: Mon Mar 16 14:20:45 2009

Total megahubs: 0
We have 50442493 skims in file.
Loading skims in id range 0 to 11434885
Loaded 89478494 elements.
Loading skims in id range 11434885 to 14819703
Loaded 11406492 elements.
Loading skims in id range 0 to 11434885
Loaded 89478494 elements.
Loading skims in id range 11434885 to 14819703
Loaded 11406492 elements.
Loading skims in id range 0 to 11434885
Loaded 89478494 elements.
Loading skims in id range 11434885 to 14819703
Loaded 11406492 elements.
Total skims taken: 12933262

Filtering forward skims.
Localtime: Mon Mar 16 14:26:36 2009
Writing reduced skim file:
 [0%] ....|.... [10%] ....|.... [20%] ....|.... [30%] ....|.... [40%] ....|.... 
[50%] ....|.... [60%] ....|.... [70%] ....|.... [80%] ....|.... [90%] ....|.... 
[100%] 
Done.
Done.
Filtering complement skims.
Localtime: Mon Mar 16 14:26:45 2009
Writing reduced skim file:
 [0%] ....|.... [10%] ....|.... [20%] ....|.... [30%] ....|.... [40%] ....|.... 
[50%] ....|.... [60%] ....|.... [70%] ....|.... [80%] ....|.... [90%] ....|.... 
[100%] 
Done.
Done.


Pass: 1

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