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-   -   FASTQ to SAM conversion (http://seqanswers.com/forums/showthread.php?t=5325)

kurban910 08-07-2014 06:42 AM

commend in terminal:
kurban@kurban-X550VC:~/Downloads/bwa-0.7.10$ bwa mem gene.fa CD_ATGTCA_L007_R1_001.fastq CD_ATGTCA_L007_R1_002.fastq > aln-pe1.sam
and it shows:
[main] unrecognized command 'mem'

is this a problem of bwa version or what? any suggestion would be appreciated.

maubp 08-07-2014 07:02 AM

Probably - which version of bwa are you using? Note your command did NOT run a local copy of bwa in the current folder, but the system default via the $PATH setting.

Brian Bushnell 08-07-2014 08:40 AM

Adding a "./" might fix it, if the bwa executable is in that directory.
./bwa mem gene.fa CD_ATGTCA_L007_R1_001.fastq CD_ATGTCA_L007_R1_002.fastq > aln-pe1.sam

kurban910 08-07-2014 10:02 AM

thank you guys, it seems like a problem of old version bwa. but i have another question:
i have a five pairs of raw reads files all in fastq format as blow:
CD_ATGTCA_L007_R1_001.fastq CD_ATGTCA_L007_R2_004.fastq
CD_ATGTCA_L007_R1_002.fastq CD_ATGTCA_L007_R2_005.fastq
CD_ATGTCA_L007_R1_003.fastq
CD_ATGTCA_L007_R1_004.fastq
CD_ATGTCA_L007_R1_005.fastq
CD_ATGTCA_L007_R2_001.fastq
CD_ATGTCA_L007_R2_002.fastq
CD_ATGTCA_L007_R2_003.fastq

they are the paired end raw reads of the insects we sequenced. if i execute alignment $ bwa mem ref.fa read1.fq read2.fq > aln-pe.sam
i would get five sam files, right? but along the way of SNPs calling should i add these files into one whole file? if i do ,which step should i do that and how?

Brian Bushnell 08-07-2014 10:11 AM

If they are all the same library, I would cat them first.
cat CD_ATGTCA_L007_R1_*.fastq > r1.fq
cat CD_ATGTCA_L007_R2_*.fastq > r2.fq

Then map. You could concatenate them after mapping, also, if you strip the headers, but this is simpler.

GenoMax 08-07-2014 10:11 AM

You should "cat" the R1 and R2 reads together into a single file. Then use that file to do the alignments. Keep the order of the files intact (1 --> 5) as you cat them.

kurban910 08-07-2014 10:29 AM

thank you guys , i really learned a lot. but it's already midnight here,so i would do that first thing in the morning, c u.

kurban910 08-08-2014 03:25 AM

hello!
when i tried to filter SNPs by typing the commend in the terminal:
bcftools view my.var.bcf | vcfutils.pl varFilter - > my.var-final.vcf

it showed this:
open: No such file or directory
vcfutils.pl: command not found

then i found the location of vcfutils.pl :
$ locate vcfutils.pl
/usr/share/samtools/vcfutils.pl

then i typed the commend in terminal:
$ bcftools view my.var.bcf | /usr/share/samtools/vcfutils.pl varFilter - > my.var-final.vcf
it still gives me this:
open: No such file or directory

and i checked the file vcfutils.pl ,it there. i even put the commend like this way:
$ bcftools view my.var.bcf | perl /usr/share/samtools/vcfutils.pl varFilter - > my.var-final.vcf
it still gived me this:
open: No such file or directory

where should i make change in the command line this time?

GenoMax 08-08-2014 03:45 AM

Is the bcftools executable in the directory you are currently in? Have you tried to "locate" it and provide the full path for it like you did for vcfutils.pl?

Is your my.var.bcf file in the current directory?

In future, start a new thread when you have a new question.

blakeoft 08-08-2014 03:49 AM

kurban910, are you sure that you have the right name for the bcf file? It looks to me like the command is correct.

kurban910 08-08-2014 04:18 AM

Quote:

Originally Posted by GenoMax (Post 147102)
Is the bcftools executable in the directory you are currently in? Have you tried to "locate" it and provide the full path for it like you did for vcfutils.pl?

Is your my.var.bcf file in the current directory?

In future, start a new thread when you have a new question.

1. bcftools is executable in my current directory.

kurban@kurban-X550VC:~/Desktop/SNPs/CD$ bcftools

Program: bcftools (Tools for data in the VCF/BCF formats)
Version: 0.1.17-dev (r973:277)

Usage: bcftools <command> <arguments>

Command: view print, extract, convert and call SNPs from BCF
index index BCF
cat concatenate BCFs
ld compute all-pair r^2
ldpair compute r^2 between requested pairs


2.i have tried to provide full path of bcftools:
kurban@kurban-X550VC:~/Desktop/SNPs/CD$ /usr/bin/bcftools view my.var.bcf | /usr/share/samtools/vcfutils.pl varFilter - > my.var-final.vcf
it says:
open: No such file or directory
and file my.var.bcf is in my current diractory.

but it is still not working.

GenoMax 08-08-2014 04:27 AM

Are you sure bcftools in in your current directory (~/Desktop/SNPs/CD from what I can see above)? You seem to be running the copy that is in /usr/bin (in the set of commands in #2).

Have you verified that my.var.bcf file is non-zero bytes (i.e. there is something in it)?

Can you use [ CODE] (remove the leading space before CODE) put your commands here [/CODE] to make the commands you are pasting clear (enclose them in CODE tags on both sides like I have shown). Otherwise it is difficult to decipher if there are spaces in wrong spot in your command line.

blakeoft 08-08-2014 04:27 AM

kurban910, I just ran
Code:

bcftools view jim.bcf
and I don't have any file called jim.bcf. It gave me this:
Code:

open: No such file or directory
This makes me think that you have the wrong name. Be careful with 1's and l's, -'s and _'s, etc -- it's easy to get these confused. Heck, I even get .'s and _'s confused sometimes. In the directory that the bcf file is located, can you execute:
Code:

ls $PWD/*.bcf
and copy the bcf file's name exactly as it appears, including the full path, and then try running your command again?

kurban910 08-08-2014 04:37 AM

Quote:

Originally Posted by blakeoft (Post 147103)
kurban910, are you sure that you have the right name for the bcf file? It looks to me like the command is correct.

yes, i am sure the file name is right:(

kurban910 08-08-2014 04:44 AM

Quote:

Originally Posted by GenoMax (Post 147107)
Are you sure bcftools in in your current directory (~/Desktop/SNPs/CD from what I can see above)? You seem to be running the copy that is in /usr/bin (in the set of commands in #2).

Have you verified that my.var.bcf file is non-zero bytes (i.e. there is something in it)?

Can you use [ CODE] (remove the leading space before CODE) put your commands here [/CODE] to make the commands you are pasting clear (enclose them in CODE tags on both sides like I have shown). Otherwise it is difficult to decipher if there are spaces in wrong spot in your command line.

u r right ,thanks. i will put the commends in the code box next time. and yes, my.var.bcf file is around 7.6 BM.

and for your first question, bcftools is not in the current directory i am in, but its executable from here.

kurban910 08-08-2014 05:28 AM

Quote:

Originally Posted by blakeoft (Post 147108)
kurban910, I just ran
Code:

bcftools view jim.bcf
and I don't have any file called jim.bcf. It gave me this:
Code:

open: No such file or directory
This makes me think that you have the wrong name. Be careful with 1's and l's, -'s and _'s, etc -- it's easy to get these confused. Heck, I even get .'s and _'s confused sometimes. In the directory that the bcf file is located, can you execute:
Code:

ls $PWD/*.bcf
and copy the bcf file's name exactly as it appears, including the full path, and then try running your command again?

thank you for your time guys , now i find out not just this one, other commends also r not exacted in my terminal , ubuntu 12.04 some times isnít stable . after i reinstall the system i will try again.

maubp 09-18-2015 07:04 AM

Quote:

Originally Posted by maubp (Post 19694)
If you did want to convert the FASTQ to an unaligned SAM or BAM file, try this:
http://picard.sourceforge.net/comman...tml#FastqToSam

I need to do this locally, and since we didn't have Picard installed but do have Biopython (yes, I'm biased ;) ), I wrote a simple Python script to convert paired FASTQ files into unmapped SAM reads (which you can pipe into samtools to get as a BAM file):

https://github.com/peterjc/picobio/b...astq_to_sam.py

I would expect this to be slower than a dedicated tool, so probably not suitable for a high throughput pipeline - but it should be fine for a one-off analysis.


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