Hello, I am having trouble replicating the great output you get after running samtools "Filter pileup on coverage & snps with ten columns (with consensus)" when I try to run samtools locally on my computer. Unfortunately we are unable to use Galaxy with our new data as our files are too large to upload to the website. Do you use some other scripts in the back ground to get such an informative output? When I run the samtools commands samtools pileup -i -vcf RefSeq.fa aln_sorted.bam > aln_ivcf.pileup and then samtools.pl varFilter aln_ivcf.pileup | awk '($3=="*" && $6>=20 && $7>=20 && $8>=10)' > final_aln_ivcf.pileup I do not get useful information in the output that tells me how many reads are calling the alternative allele, & what the alternative allele is. Any help would be gratefully received. Thank you Alison Gardner