[WhatsOEver]

Since you mention SNP calling, you'll want to use a tools like BWA or bowtie2 rather than tophat for alignment. Aside from that, I'm in agreement with WhatsOEver.

Agreed
I mentioned tophat as it has imo the best documentation and is therefore the easiest to start with. But in the end, it might not be the best solution here.

[kurban910]

i am glad for you guys reply, thanks.

[kurban910]

hello guys!
i have downloaded bwa-0.7.10, and then uncompressed it, then
kurban@kurban-X550VC:~/Downloads/bwa-0.7.10$ make

it showed this :
gcc -c -g -Wall -Wno-unused-function -O2 -DHAVE_PTHREAD -DUSE_MALLOC_WRAPPERS utils.c -o utils.o
utils.c:33:18: fatal error: zlib.h: No such file or directory
compilation terminated.
make: *** [utils.o] Error 1
what is what is happening there ?

[maubp]

You (or your SysAdmin) need to install the zlib library including the development files (header files like zlib.h).

[kurban910]

thank you, after installing zlib it worked

[kurban910]

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]

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]

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]

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]

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]

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]

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]

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]

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]

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