Hi all,
I am new to NGS analysis. I have used bowtie (ver:bowtie-0.12.7) for aligning reference sequence (fastq format) with two paired end files of illumina reads (fastq format).
I used SAM tools (ver:samtools-0.1.18) and made a 'mpileup' file.
Then I have used Varscan (ver:.v2.2.11) for variant calling. I used "mpileup2snp" command (with default parameters) to determine SNV and for heterozygosity & homozygosity.
My basic idea is to call heterozygous SNV with conservative filters, such as coverage, variant allele
frequency, strand representation and p-value to isolate high-confidence calls.
Using the command "mpileup2snp", I got the output as follows. Given 1 column as an example
Chrom - gi|371443199|gb|JH556661.1|
Position - 2594265
Ref- G
Var- C
Cons:Cov:Reads1:Reads2:Freq:P-value- S:21:14:7:33.33%:4.3101E-3
StrandFilter:R1+:R1-:R2+:R2-
val- Fail:12:2:0:7:3.096E-4
SamplesRef- 0
SamplesHet- 1
SamplesHom- 0
SamplesNC - 0
Cons:Cov:Reads1:Reads2:Freq:P-value- S:21:14:7:33.33%:4.3101E-3
1.Please let me know whether I am using the correct command to call heterzygous SNV with high-confidence calls(whether I have to use mpileup2snp or mpileup2indel or mpileup2cns?)
2.Can I parse these variant call by chromosome?. How Can I do that?
3.Is it possible to view regions of high and low heterozygosity in the genome?. If possible, how can I do that?
Thanks in advance
I am new to NGS analysis. I have used bowtie (ver:bowtie-0.12.7) for aligning reference sequence (fastq format) with two paired end files of illumina reads (fastq format).
I used SAM tools (ver:samtools-0.1.18) and made a 'mpileup' file.
Then I have used Varscan (ver:.v2.2.11) for variant calling. I used "mpileup2snp" command (with default parameters) to determine SNV and for heterozygosity & homozygosity.
My basic idea is to call heterozygous SNV with conservative filters, such as coverage, variant allele
frequency, strand representation and p-value to isolate high-confidence calls.
Using the command "mpileup2snp", I got the output as follows. Given 1 column as an example
Chrom - gi|371443199|gb|JH556661.1|
Position - 2594265
Ref- G
Var- C
Cons:Cov:Reads1:Reads2:Freq:P-value- S:21:14:7:33.33%:4.3101E-3
StrandFilter:R1+:R1-:R2+:R2-
val- Fail:12:2:0:7:3.096E-4 SamplesRef- 0
SamplesHet- 1
SamplesHom- 0
SamplesNC - 0
Cons:Cov:Reads1:Reads2:Freq:P-value- S:21:14:7:33.33%:4.3101E-3
1.Please let me know whether I am using the correct command to call heterzygous SNV with high-confidence calls(whether I have to use mpileup2snp or mpileup2indel or mpileup2cns?)
2.Can I parse these variant call by chromosome?. How Can I do that?
3.Is it possible to view regions of high and low heterozygosity in the genome?. If possible, how can I do that?
Thanks in advance