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**GenoMax** · 11-03-2014, 06:44 AM

The command line you are using refers to the older version of bcftools (v.0.1.19). The -b option is deprecated in new bcftools (v.1.1), which you are using.

I have not tested the following command line. See if it works for you.

Code:

$ samtools mpileup -uf /example/reference/lambda_virus.fa eg2.sorted.bam | bcftools view -O u -v -c 5 -g hom - > eg2.raw.bcf

PS: If you type

Code:

$ bcftools view

Then that should print the help for the bcftools view command. You can then choose the appropriate command line options for various parameters.

**zillur** · 11-03-2014, 06:51 AM

Thank you very much.

Mds-MacBook-Pro:reference ZILLURRAHMAN$ ls
commands lambda_virus.3.bt2 lambdaout.4.bt2
eg2.bam lambda_virus.4.bt2 lambdaout.rev.1.bt2
eg2.raw.bcf lambda_virus.fa lambdaout.rev.2.bt2
eg2.sam lambda_virus.rev.1.bt2 nohup.out
eg2.sorted.bam lambda_virus.rev.2.bt2 out
egl.sam lambdaout.1.bt2 out.sam
lambda_virus.1.bt2 lambdaout.2.bt2
lambda_virus.2.bt2 lambdaout.3.bt2
Mds-MacBook-Pro:reference ZILLURRAHMAN$ samtools mpileup -uf /example/reference/lambda_virus.fa eg2.sorted.bam | bcftools view -O u -v -c 5 -g hom - > eg2.raw.bcf
[fai_load] build FASTA index.
[fai_build] fail to open the FASTA file /example/reference/lambda_virus.fa
[fai_load] fail to open FASTA index.
[E::hts_open] fail to open file '5'
Failed to open or the file not indexed: 5
Mds-MacBook-Pro:reference ZILLURRAHMAN$

Which thing I am missing?

Best Regards
Zillur

**GenoMax** · 11-03-2014, 07:08 AM

This should work.

Code:

$ samtools mpileup -uf /example/reference/lambda_virus.fa eg2.sorted.bam | bcftools view -O u  - > eg2.raw.bcf

**zillur** · 11-03-2014, 07:13 AM

Thank you very much for your patience.

Mds-MacBook-Pro:reference ZILLURRAHMAN$ samtools mpileup -uf /example/reference/lambda_virus.fa eg2.sorted.bam | bcftools view -O u - > eg2.raw.bcf
[fai_load] build FASTA index.
[fai_build] fail to open the FASTA file /example/reference/lambda_virus.fa
[fai_load] fail to open FASTA index.
[vcf.c:1224 vcf_hdr_read] Could not read the header
Failed to open or the file not indexed: -
Mds-MacBook-Pro:reference ZILLURRAHMAN$ ls
commands lambda_virus.3.bt2 lambdaout.4.bt2
eg2.bam lambda_virus.4.bt2 lambdaout.rev.1.bt2
eg2.raw.bcf lambda_virus.fa lambdaout.rev.2.bt2
eg2.sam lambda_virus.rev.1.bt2 nohup.out
eg2.sorted.bam lambda_virus.rev.2.bt2 out
egl.sam lambdaout.1.bt2 out.sam
lambda_virus.1.bt2 lambdaout.2.bt2
lambda_virus.2.bt2 lambdaout.3.bt2
Mds-MacBook-Pro:reference ZILLURRAHMAN$

May be I am missing some points.

**GenoMax** · 11-03-2014, 07:19 AM

The command should be (I was going off what you had in your prior post)

Code:

$ samtools mpileup -uf $BT2_HOME/example/reference/lambda_virus.fa eg2.sorted.bam | bcftools view -O u - > eg2.raw.bcf

**zillur** · 11-03-2014, 07:37 AM

Thank you very much. I think its working now.
But what does it mean?

Mds-MacBook-Pro:reference ZILLURRAHMAN$ samtools mpileup -uf lambda_virus.fa eg2.sorted.bam | bcftools view -O u - > eg2.raw.bcf
[fai_load] build FASTA index.
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000
Mds-MacBook-Pro:reference ZILLURRAHMAN$ ls
commands lambda_virus.3.bt2 lambdaout.3.bt2
eg2.bam lambda_virus.4.bt2 lambdaout.4.bt2
eg2.raw.bcf lambda_virus.fa lambdaout.rev.1.bt2
eg2.sam lambda_virus.fa.fai lambdaout.rev.2.bt2
eg2.sorted.bam lambda_virus.rev.1.bt2 nohup.out
egl.sam lambda_virus.rev.2.bt2 out
lambda_virus.1.bt2 lambdaout.1.bt2 out.sam
lambda_virus.2.bt2 lambdaout.2.bt2
Mds-MacBook-Pro:reference ZILLURRAHMAN$ more lambda_virus.fa.fai
gi|9626243|ref|NC_001416.1| 48502 74 70 71
Mds-MacBook-Pro:reference ZILLURRAHMAN$ head lambda_virus.fa.fai
gi|9626243|ref|NC_001416.1| 48502 74 70 71
Mds-MacBook-Pro:reference ZILLURRAHMAN$

**GenoMax** · 11-03-2014, 07:56 AM

What it means is you just used a data file containing alignments against lambda virus and used that to call SNP's (bcftools part). Last command will let you look at the SNP's called.

Code:

$ bcftools view eg2.raw.bcf

**zillur** · 11-03-2014, 08:25 AM

Thank you very much. Now I got large data like:

gi|9626243|ref|NC_001416.1| 48491 . C <X> 0 . DP=6;I16=0,3,0,0,84,2570,0,0,87,3537,0,0,25,625,0,0;QS=1,0;MQ0F=0 PL 0,9,47
gi|9626243|ref|NC_001416.1| 48492 . G <X> 0 . DP=4;I16=0,3,0,0,73,1801,0,0,87,3537,0,0,29,635,0,0;QS=1,0;MQ0F=0 PL 0,9,42
gi|9626243|ref|NC_001416.1| 48493 . A <X> 0 . DP=4;I16=0,3,0,0,72,1910,0,0,87,3537,0,0,31,661,0,0;QS=1,0;MQ0F=0 PL 0,9,50
gi|9626243|ref|NC_001416.1| 48494 . C <X> 0 . DP=3;I16=0,3,0,0,80,2194,0,0,87,3537,0,0,31,651,0,0;QS=1,0;MQ0F=0 PL 0,9,45
gi|9626243|ref|NC_001416.1| 48495 . A <X> 0 . DP=3;I16=0,2,0,0,43,1037,0,0,45,1773,0,0,25,625,0,0;QS=1,0;MQ0F=0 PL 0,6,16
gi|9626243|ref|NC_001416.1| 48496 . G <X> 0 . DP=2;I16=0,2,0,0,65,2125,0,0,45,1773,0,0,28,634,0,0;QS=1,0;MQ0F=0 PL 0,6,37
gi|9626243|ref|NC_001416.1| 48497 . G <X> 0 . DP=2;I16=0,1,0,0,27,729,0,0,3,9,0,0,25,625,0,0;QS=1,0;MQ0F=0 PL 0,3,4
gi|9626243|ref|NC_001416.1| 48498 . T <X> 0 . DP=2;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0
gi|9626243|ref|NC_001416.1| 48499 . T <X> 0 . DP=2;I16=0,1,0,0,33,1089,0,0,42,1764,0,0,0,0,0,0;QS=1,0;MQ0F=0 PL 0,3,33
gi|9626243|ref|NC_001416.1| 48500 . A <X> 0 . DP=1;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0
gi|9626243|ref|NC_001416.1| 48501 . C <X> 0 . DP=1;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0
gi|9626243|ref|NC_001416.1| 48502 . G <X> 0 . DP=1;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0

so many of this. How can I interpret this data?

Best Regards
Zillur

**zillur** · 11-04-2014, 05:04 AM

[QUOTE=GenoMax;153671]This is a simple work around.

Since you are having trouble editing .bash_profile file put the PATH command in a text file (let us call the file "set_path_file") using an editor. Then when you open a new terminal do the following

Code:

$ source set_path_file

Thank you very much. Using an editor I created a text file where I set the path for bowtie2, smalls, bcftools. Then in a new terminal session I typed "$ source path command.txt" it seems working and direct me to the parent directory. But commands are not found.

Mds-MacBook-Pro:~ ZILLURRAHMAN$ type bowtie
-bash: type: bowtie: not found

I have attached the file so that you can guess my mistakes.

Best Regards
Zillur

Attached Files

pathcommand.txt (281 Bytes, 1 view)

**GenoMax** · 11-04-2014, 05:09 AM

You need to stop at the level of the directory (not include the actual executable). We are letting the system know that the file(s) I am looking for are in this directory path.

Modified file attached.

Attached Files

pathcommand_mod.txt (206 Bytes, 105 views)

**zillur** · 11-04-2014, 05:16 AM

Thank you very much. Its working now.
another issue, using $ bcftools view eg2.raw.bcf I have got a large data containing like these:

gi|9626243|ref|NC_001416.1| 48496 . G <X> 0 . DP=2;I16=0,2,0,0,65,2125,0,0,45,1773,0,0,28,634,0,0;QS=1,0;MQ0F=0 PL 0,6,37
gi|9626243|ref|NC_001416.1| 48497 . G <X> 0 . DP=2;I16=0,1,0,0,27,729,0,0,3,9,0,0,25,625,0,0;QS=1,0;MQ0F=0 PL 0,3,4
gi|9626243|ref|NC_001416.1| 48498 . T <X> 0 . DP=2;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0
gi|9626243|ref|NC_001416.1| 48499 . T <X> 0 . DP=2;I16=0,1,0,0,33,1089,0,0,42,1764,0,0,0,0,0,0;QS=1,0;MQ0F=0 PL 0,3,33
gi|9626243|ref|NC_001416.1| 48500 . A <X> 0 . DP=1;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0
gi|9626243|ref|NC_001416.1| 48501 . C <X> 0 . DP=1;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0
gi|9626243|ref|NC_001416.1| 48502 . G <X> 0 . DP=1;I16=0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0;QS=0,0;MQ0F=0 PL 0,0,0

so many of this. How can I interpret this data?

Best Regards
Zillur

**GenoMax** · 11-04-2014, 06:20 AM

That file is in VCF v.4.2 format. You can find the details about the format here: http://samtools.github.io/hts-specs/VCFv4.2.pdf

**zillur** · 11-04-2014, 06:59 AM

Thank you very much. I am working on it. I will inform you about my progresses. Thank you again.

Best Regards
Zillur

**zillur** · 11-05-2014, 09:12 AM

Now I have to measure the quality of the data that we generated as vcc format. Would you please to give me some suggestions by which I can measure the quality of the data.

Best Regards
Zillur

**GenoMax** · 11-05-2014, 09:28 AM

Originally posted by zillur View Post

Now I have to measure the quality of the data that we generated as vcc format. Would you please to give me some suggestions by which I can measure the quality of the data.

Best Regards
Zillur

Since this question is no longer associated with the subject of this thread you should post it as a separate question (after due diligence of searching SeqAnswers.com for prior postings).

To start a new thread: http://seqanswers.com/forums/showpos...69&postcount=9

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