yes seems like it..
So i just wanted to ask you if appending the shotgun files is a must ?
have you done the appending and performed the assembly ?
If NO, what are the softwares u suggest I do a 454 denovo with ?
Since you converted to fastq, i assume you used an illumina assembler ?
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It looks like you don't have enough memory to do this assembly. As mentioned before, try the miramem command to estimate the memory requirement for this assembly....that way you'll know if you're in the ball park of what is required by MIRA.
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The log file at the end showed this :
Dynamic allocs: 0
Align allocs: 0
Out of memory detected, exception message is: std::bad_alloc
You are running a 32 bit executable. Please note that the maximum
theoretical memory a 32 bit programm can use (be it in Linux, Windows or
other) is 4 GiB, in practice less: between 2.7 and 3.3 GiB. This is valid
even if your machine has hundreds of GiB.
Should your machine have more that 4 GiB, use a 64 bit OS and a 64 bit
version of MIRA.
----
So i downloaded the 64 bit of MIRA and after the command
mira --project=3kb_norton --job=denovo,genome,accurate,454 -SK:mnr=yes:nrr=10 >&3kb_log_assembly.txt
it said:
tcmalloc: large alloc 2323759104 bytes == 0*1f5b9000 @
so the number of bytes increased
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Extracting the files from sff:
sff_extract -c -l linker.fasta "insert_size:2500,insert_stdev:500" file1.sff file2.sff -o 3kb_norton
This worked perfect and had given me the fasta, qual and xml..
Appending shotgun files:
sff_extract -a shotgun1.sff shotgun2.sff shotgun3.sff -o 3kb_norton
This also appended the sff's and the sizes of the initial fasta,qual and xml files changed to a much larger size to allocate the shotgun seqs...
Assembly:
mira --project=3kb_norton --job=denovo,genome,accurate,454 -SK:mnr=yes:nrr=10 >&3kb_log_assembly.txt
now this also showed no error.. it started running.. and after a while below the command it said:
tcmalloc: large alloc 1482399744 bytes == 0*867e000 @
and below this, after a while it said:
Aborted
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Originally posted by aarthi.talla View PostThank you
Do we have to perform the step of appending the shotgun files to the extracted paried end fasta files ? Or can we do the assembly of the extracted files by sff extract directly ?
Because when I appended the shotgun files to the extracted paired end fasta files and performed the assembly , it shows memory allocation problem !! The memory of my linux machine is 7GB.. isnt that enough ? how much memory does mira require to perform the asembly ?
may I know how u performed your assembly ? did u append the shotgun files or just did an assembly of the paired ends extracted fasta files ??
Thanks
MIRA is an Overlap/Layout/Consensus (OLC) type assembler. They inherently require lots of memory for all but the smallest genomes. Try using the miramem command to estimate what the memory requirement is likely to be.
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have you used MIRA to perform assembly ? If NO, then which wud u suggest ? Since you have converted the sff's to fastq, i assume you used an illumina denovo software??
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Thank you
Do we have to perform the step of appending the shotgun files to the extracted paried end fasta files ? Or can we do the assembly of the extracted files by sff extract directly ?
Because when I appended the shotgun files to the extracted paired end fasta files and performed the assembly , it shows memory allocation problem !! The memory of my linux machine is 7GB.. isnt that enough ? how much memory does mira require to perform the asembly ?
may I know how u performed your assembly ? did u append the shotgun files or just did an assembly of the paired ends extracted fasta files ??
Thanks
Leave a comment:
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Originally posted by aarthi.talla View PostThankyou very much ! that was really helpful.
I am sorry to bother you with all the questions.
Can I ask you one last question.
Originally posted by aarthi.talla View PostFor the scaffolding with bambus is it necessary that we provide the mates file ?
Originally posted by aarthi.talla View PostIf yes, since we cannot read the sff file, do all the sff's contain the format that i mentioned ? (.*)\.f (.*)\.r (with an 'f' and and 'r' to it) ? And can I just blindly assume to give in this ??
may I know if you have provided the mates and the conf file ?
Thanks !!
e.g. you would have a simplified workflow something like this:
Code:file.sff ----> sff_extract ----> file.fastq ----> chosen_assembly_tool ----> assembly_output
Here's some more resources you may find useful:
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Thankyou very much ! that was really helpful.
I am sorry to bother you with all the questions.
Can I ask you one last question.
For the scaffolding with bambus is it necessary that we provide the mates file ?
If yes, since we cannot read the sff file, do all the sff's contain the format that i mentioned ? (.*)\.f (.*)\.r (with an 'f' and and 'r' to it) ? And can I just blindly assume to give in this ??
may I know if you have provided the mates and the conf file ?
Thanks !!
Leave a comment:
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Originally posted by aarthi.talla View PostAlso for the mates.file as I am required to provide the minimum insert size(which is mean of insert size-stddev) and maximum insert size(which is mean of insert size-stddev). I hope I got these right ?
So for example to consider the mean insert size of the 3kb run, would it just be 3000 or would it be the average of the numbers 2247.3 and 2254.9 of the 2 sff files that I mentioned earlier ??
Same is applied for the standard deviations. Do i again consider the averages ? (561.8+563.7/2) ??
Did you setup a mates files yet for scaffolding ? If yes may I know how u set it up with respect to the naming convention?
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Originally posted by aarthi.talla View PostAnd I would like to add about using shotgun sff files in the bambus scaffolding step.
Please let me know if I got this right.
When the sheared DNA fragments are circularized with an adaptor/linker, they are fragmented again. And some of these fragments will have the adaptor flanked by read pairs approx 150bp on each side, and there will be some other fragments with NO adaptor in between them obviously. So these frags with no adaptors are the shotgun sequences ?
Which is why you provide the shotgun sequence sff files to bambus so that it will not miss out on that data ?
Did I get it correct ?
Some links you might find useful
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Originally posted by aarthi.talla View PostBut they had done an initial assembly with newbler for us and in the newbler metrics, in the paired read status section a 'pairDistanceAvg' is given. So is that the insert size ?
Originally posted by aarthi.talla View Postfor the 3kb library for sff file 1 - the pairdistanceavg = 2247.3, pairDistdev=561.8 and for sff file 2 pairdistavg is 2254.9 and pairDistdev 563.7.
Why are these not 3kb ? And to enter the stddev, do i sum up both of them or do I take the average ??
Originally posted by aarthi.talla View PostAnd since they are 2 sff files per run, for 'sff_extract' can I give in the 2 sff files as input along with the script u mentioned above and will it output the fasta, qual and xml files into just one file ??
Some links you, or readers of this post, might find useful:- Newbler Blog
- Traceinfo Documentation
- sff_extract
- MIRA's 454 Assembly Docs - The author of MIRA is the author of sff_extract, so this is some good info in these docs.
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Also for the mates.file as I am required to provide the minimum insert size(which is mean of insert size-stddev) and maximum insert size(which is mean of insert size-stddev). I hope I got these right ?
So for example to consider the mean insert size of the 3kb run, would it just be 3000 or would it be the average of the numbers 2247.3 and 2254.9 of the 2 sff files that I mentioned earlier ??
Same is applied for the standard deviations. Do i again consider the averages ? (561.8+563.7/2) ??
Did you setup a mates files yet for scaffolding ? If yes may I know how u set it up with respect to the naming convention?
Leave a comment:
-
And I would like to add about using shotgun sff files in the bambus scaffolding step.
Please let me know if I got this right.
When the sheared DNA fragments are circularized with an adaptor/linker, they are fragmented again. And some of these fragments will have the adaptor flanked by read pairs approx 150bp on each side, and there will be some other fragments with NO adaptor in between them obviously. So these frags with no adaptors are the shotgun sequences ?
Which is why you provide the shotgun sequence sff files to bambus so that it will not miss out on that data ?
Did I get it correct ?
Leave a comment:
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Thank you so much
A comapany called 'Seqwright' sequenced the data for us and provided with 3kb, 8kb and 20kb libraries. So now i understand that the insert size is 3kb, 8kb and 20kb respectively.
But they had done an initial assembly with newbler for us and in the newbler metrics, in the paired read status section a 'pairDistanceAvg' is given. So is that the insert size ?
They have given 2 sff files per library, since they say- 'reason you have two files per run is because it’s sequenced on the DNA chip with two regions'.
e.g for the 3kb library for sff file 1 - the pairdistanceavg = 2247.3, pairDistdev=561.8 and for sff file 2 pairdistavg is 2254.9 and pairDistdev 563.7.
Why are these not 3kb ? And to enter the stddev, do i sum up both of them or do I take the average ??
And since they are 2 sff files per run, for 'sff_extract' can I give in the 2 sff files as input along with the script u mentioned above and will it output the fasta, qual and xml files into just one file ??
Leave a comment:
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