output samtools.pl

[m_elena_bioinfo]

Dear users,
I used BFAST program to generate a file .sam (using microRNA reads from SOLiD ABI).
I run SAMTOOLS to analyse SNPs and mutations, and I generated at the end two output files:
a pileup file and a file .snps (by samtools.pl varFilter).

This is an example of my file .snps:

g hsa-let-7a-3 61 * */+A 73 73 40 81 * +A 79 2 0 0 0
g hsa-let-7a-3 63 * */-C 133 133 45 79 * -C 76 1 2 3 1
g hsa-let-7a-3 64 * */-T 165 165 44 78 * -T 77 1 0 0 0
g hsa-let-7a-3 68 * */-T 385 385 40 53 * -T 52 1 0 0 0
g hsa-let-7a-3 70 * */+A 78 78 35 43 * +A 42 1 0 0 0
D hsa-let-7b 22 * */+A 895 895 22 1717 * +A 1652 36 29 7 37
D hsa-let-7b 24 * */+TC 14761 14761 23 1728 * +TC 1573 51 104 7 4
D hsa-let-7b 25 * +TA/+CTA 17955 37444 23 1730 +TA +CTA 32 17 1681 35 18
D hsa-let-7b 26 * +A/+TA 21497 46739 23 1732 +A +TA 393 162 1177 40 29
D hsa-let-7b 27 * +A/* 31077 31077 23 1733 +A * 6 1651 76 4 5
D hsa-let-7b 28 T Y 228 228 23 1736 ,,.+1C,,$C.C...+4ATTC.+4ATTC*.+4ATTCC,.+1C.,...+4ATTCC..+1CCC.+1CC.+4ATTC
.+1C.+4ATTC.+1C..+4ATTC.+4ATTC.+4ATTCC.+1C*.+4ATTCC.+1CC.+4ATTCC.+1C...+1CCC.+1C.+4ATTCC..+1CC.+1C.+1C.+1C.+1CC.+1C.+4ATTCC.+1C.+1C...+4ATTC.+1C.C..+1C.C
..+1C..+1C..+1C.....+4ATTCC.+4ATTCC..+4ATTC..C.+4ATTC.C.+1C.+4ATTCC.+1C.+4ATTCC...+4ATTC.C.+4ATTC.+1C......C.+4ATTC..+1C.+4ATTC.+1C.C.+4ATTC.+1CCCC.+1C.+
1C.+4ATTC.+1C.+4ATTC.+1C.+4ATTC.+4ATTC.+4ATTC.+1C.+1CC.+4ATTC.+1CCC.+1CC.+1C.+1CC.C.+1CC.+1C.+1C.+4ATTC.+4ATTC..+1C.+1C.+1CCC.+4ATTCC.+1CC.+1C...C.+1C.+1
CC.+4ATTC..+1C.+1C.+1C.+1C*.+4ATTC..+4ATTC.+1C.+1C.+1C.+1C.+1CC.CC.+4ATTC.+1C.+1CC..+1C..+1C.+1C..+1C.+4ATTC.+1C.+1C.+1C...+1C...+1C.A.+4ATTC.+4ATTC.-1C.
+3CGC.+2TC.+1C.+1CC.+1C.+4ATTC..+4ATTC.+4ATTCC..+1C.+1C.C.CC.+4ATTCC.+4ATTC.+1C.+1C.+4ATTC.+1CC.C.+1C.+4ATTC.+4ATTC.+4ATTC.+1CC.+4ATTCC.+4ATTC.+4ATTC.+1C
.+1CCC.+1C.+1C.+1C.+1C.+1CC.+4ATTC.+1CCC.+1CCCC.+1C.+1C.+4ATTC.+1CC.+1C..+4ATTC.CC.+1C.+4ATTC.+1C.CC.+1C.C.+1C.+1C.+1CC.+1CC.+1C.+1C.+1C.+4ATTC.+4ATTC.+1
C.+4ATTCC.+1C.+1C.+1C.+1C.+1CC.+5CATTC.+4ATTC.+4ATTCCC.+1C.+1C.+1CC..C.+4ATTC.+1C..+1C..+1C.+1CC.+1C*.+1CCC.+4ATTC.+4ATTC..+1C.+1C..+1C..C.+4ATTC.+4ATTC.
+4ATTC.C.+4ATTC.+1CC..+1C.+1CC.+4ATTC.+1C.+1C.CCC.+1C.+1C.+4ATTC.+4ATTC.+1C.+1CC..-1CC.+1C.+1C.+1C..+1C.+1C..+1C*...+1CC.+1C...+4ATTC.+1C.+1CC..+4ATTC..+
1C...+1CC.+1C.+1C..C.+1C.-2CACC..C..+4ATTCA.+1C..+1C.+4ATTC..+1C.+1C..+1CC.+4ATTC.+4ATTC.+1C.+1C..+6GTATTCC.C.+4ATTC.+1C..+1C.+4ATTC.+4ATTC.+4ATTCC.

But I don't know how I can read this file. What correspond to each column?
How or where can I find the number of reads (the number of count of miRNA in the alignment)?

Thanx a lot!
Bye
M.Elena

[nilshomer]

Dear users,
I used BFAST program to generate a file .sam (using microRNA reads from SOLiD ABI).
I run SAMTOOLS to analyse SNPs and mutations, and I generated at the end two output files:
a pileup file and a file .snps (by samtools.pl varFilter).

This is an example of my file .snps:

g hsa-let-7a-3 61 * */+A 73 73 40 81 * +A 79 2 0 0 0
g hsa-let-7a-3 63 * */-C 133 133 45 79 * -C 76 1 2 3 1
g hsa-let-7a-3 64 * */-T 165 165 44 78 * -T 77 1 0 0 0
g hsa-let-7a-3 68 * */-T 385 385 40 53 * -T 52 1 0 0 0
g hsa-let-7a-3 70 * */+A 78 78 35 43 * +A 42 1 0 0 0
D hsa-let-7b 22 * */+A 895 895 22 1717 * +A 1652 36 29 7 37
D hsa-let-7b 24 * */+TC 14761 14761 23 1728 * +TC 1573 51 104 7 4
D hsa-let-7b 25 * +TA/+CTA 17955 37444 23 1730 +TA +CTA 32 17 1681 35 18
D hsa-let-7b 26 * +A/+TA 21497 46739 23 1732 +A +TA 393 162 1177 40 29
D hsa-let-7b 27 * +A/* 31077 31077 23 1733 +A * 6 1651 76 4 5
D hsa-let-7b 28 T Y 228 228 23 1736 ,,.+1C,,$C.C...+4ATTC.+4ATTC*.+4ATTCC,.+1C.,...+4ATTCC..+1CCC.+1CC.+4ATTC
.+1C.+4ATTC.+1C..+4ATTC.+4ATTC.+4ATTCC.+1C*.+4ATTCC.+1CC.+4ATTCC.+1C...+1CCC.+1C.+4ATTCC..+1CC.+1C.+1C.+1C.+1CC.+1C.+4ATTCC.+1C.+1C...+4ATTC.+1C.C..+1C.C
..+1C..+1C..+1C.....+4ATTCC.+4ATTCC..+4ATTC..C.+4ATTC.C.+1C.+4ATTCC.+1C.+4ATTCC...+4ATTC.C.+4ATTC.+1C......C.+4ATTC..+1C.+4ATTC.+1C.C.+4ATTC.+1CCCC.+1C.+
1C.+4ATTC.+1C.+4ATTC.+1C.+4ATTC.+4ATTC.+4ATTC.+1C.+1CC.+4ATTC.+1CCC.+1CC.+1C.+1CC.C.+1CC.+1C.+1C.+4ATTC.+4ATTC..+1C.+1C.+1CCC.+4ATTCC.+1CC.+1C...C.+1C.+1
CC.+4ATTC..+1C.+1C.+1C.+1C*.+4ATTC..+4ATTC.+1C.+1C.+1C.+1C.+1CC.CC.+4ATTC.+1C.+1CC..+1C..+1C.+1C..+1C.+4ATTC.+1C.+1C.+1C...+1C...+1C.A.+4ATTC.+4ATTC.-1C.
+3CGC.+2TC.+1C.+1CC.+1C.+4ATTC..+4ATTC.+4ATTCC..+1C.+1C.C.CC.+4ATTCC.+4ATTC.+1C.+1C.+4ATTC.+1CC.C.+1C.+4ATTC.+4ATTC.+4ATTC.+1CC.+4ATTCC.+4ATTC.+4ATTC.+1C
.+1CCC.+1C.+1C.+1C.+1C.+1CC.+4ATTC.+1CCC.+1CCCC.+1C.+1C.+4ATTC.+1CC.+1C..+4ATTC.CC.+1C.+4ATTC.+1C.CC.+1C.C.+1C.+1C.+1CC.+1CC.+1C.+1C.+1C.+4ATTC.+4ATTC.+1
C.+4ATTCC.+1C.+1C.+1C.+1C.+1CC.+5CATTC.+4ATTC.+4ATTCCC.+1C.+1C.+1CC..C.+4ATTC.+1C..+1C..+1C.+1CC.+1C*.+1CCC.+4ATTC.+4ATTC..+1C.+1C..+1C..C.+4ATTC.+4ATTC.
+4ATTC.C.+4ATTC.+1CC..+1C.+1CC.+4ATTC.+1C.+1C.CCC.+1C.+1C.+4ATTC.+4ATTC.+1C.+1CC..-1CC.+1C.+1C.+1C..+1C.+1C..+1C*...+1CC.+1C...+4ATTC.+1C.+1CC..+4ATTC..+
1C...+1CC.+1C.+1C..C.+1C.-2CACC..C..+4ATTCA.+1C..+1C.+4ATTC..+1C.+1C..+1CC.+4ATTC.+4ATTC.+1C.+1C..+6GTATTCC.C.+4ATTC.+1C..+1C.+4ATTC.+4ATTC.+4ATTCC.

But I don't know how I can read this file. What correspond to each column?
How or where can I find the number of reads (the number of count of miRNA in the alignment)?

Thanx a lot!
Bye
M.Elena

I assume you are having problems understanding the samtools pileup output and BFAST ran fine. Take a look at these two pages on the samtools website that explain the output format:

http://samtools.sourceforge.net/pileup.shtml

http://samtools.sourceforge.net/cns0.shtml

[lh3]

I guess you are using "samtools.pl -p" which explains why your variants get filtered out. The first letter gives the reason:

# d low depth
# D high depth
# W too many SNPs in a window (SNP only)
# G close to a high-quality indel (SNP only)
# Q low RMS mapping quality (SNP only)
# g close to another indel with higher quality (indel only)

Note that if a SNP is filtered out due to depth, it will not be tested with "G".