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4.4 years ago
nadiabeg.comsats
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10
I have 10x reads and I need uniquely aligned reads for SNP calling. I am using samtools to extract uniquely aligned reads but I am not satisfied by the results. It would be great if someone can share the exact way of filtering these reads. I tried many combinations and checked many sources online but could not found any satisfactory answer. Here are the parameters that I already tried.
step:01 #Initial stats after alignment using freebayes:
172387804 + 0 in total (QC-passed reads + QC-failed reads)
9715270 + 0 secondary
0 + 0 supplementary
22559490 + 0 duplicates
165148332 + 0 mapped (95.80% : N/A)
162672534 + 0 paired in sequencing
81336267 + 0 read1
81336267 + 0 read2
109869698 + 0 properly paired (67.54% : N/A)
152047167 + 0 with itself and mate mapped
3385895 + 0 singletons (2.08% : N/A)
25207784 + 0 with mate mapped to a different chr
20769368 + 0 with mate mapped to a different chr (mapQ>=5)
#step:02: Picard tools was used to mark and remove duplicates
146765957 + 0 in total (QC-passed reads + QC-failed reads)
9715270 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
139526485 + 0 mapped (95.07% : N/A)
137050687 + 0 paired in sequencing
68390482 + 0 read1
68660205 + 0 read2
91962549 + 0 properly paired (67.10% : N/A)
128054904 + 0 with itself and mate mapped
1756311 + 0 singletons (1.28% : N/A)
21673676 + 0 with mate mapped to a different chr
18001625 + 0 with mate mapped to a different chr (mapQ>=5)
# To get uniquely aligned reads "parameters1:-h -q 20 -F 256 test.bam"
107311384 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
107311384 + 0 mapped (100.00% : N/A)
107311384 + 0 paired in sequencing
53437167 + 0 read1
53874217 + 0 read2
80455227 + 0 properly paired (74.97% : N/A)
105892555 + 0 with itself and mate mapped
1418829 + 0 singletons (1.32% : N/A)
15343533 + 0 with mate mapped to a different chr
15343533 + 0 with mate mapped to a different chr (mapQ>=5)
##2nd try:parameters2: -q 20 -f 0x02
82937879 + 0 in total (QC-passed reads + QC-failed reads)
2482652 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
82937879 + 0 mapped (100.00% : N/A)
80455227 + 0 paired in sequencing
40153047 + 0 read1
40302180 + 0 read2
80455227 + 0 properly paired (100.00% : N/A)
80455226 + 0 with itself and mate mapped
1 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)
#3rd try:
-f 0x02 -bq 1 (properly paired end reads)
93036316 + 0 in total (QC-passed reads + QC-failed reads)
2817262 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
93036316 + 0 mapped (100.00% : N/A)
90219054 + 0 paired in sequencing
45085486 + 0 read1
45133568 + 0 read2
90219054 + 0 properly paired (100.00% : N/A)
90219033 + 0 with itself and mate mapped
21 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)
#4th: -F 3852
105892555 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
105892555 + 0 mapped (100.00% : N/A)
105892555 + 0 paired in sequencing
52606133 + 0 read1
53286422 + 0 read2
80455226 + 0 properly paired (75.98% : N/A)
105892555 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
15343533 + 0 with mate mapped to a different chr
15343533 + 0 with mate mapped to a different chr (mapQ>=5)
#5th: -q 20 -F 268
105892555 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
105892555 + 0 mapped (100.00% : N/A)
105892555 + 0 paired in sequencing
52606133 + 0 read1
53286422 + 0 read2
80455226 + 0 properly paired (75.98% : N/A)
105892555 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
15343533 + 0 with mate mapped to a different chr
15343533 + 0 with mate mapped to a different chr (mapQ>=5)
#6th:-q 20 -F 0x100
107311384 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
107311384 + 0 mapped (100.00% : N/A)
107311384 + 0 paired in sequencing
53437167 + 0 read1
53874217 + 0 read2
80455227 + 0 properly paired (74.97% : N/A)
105892555 + 0 with itself and mate mapped
1418829 + 0 singletons (1.32% : N/A)
15343533 + 0 with mate mapped to a different chr
15343533 + 0 with mate mapped to a different chr (mapQ>=5)
Anyone suggest me the right parameter out of these tries.
I am loosing lot of reads. Is there any better way or parameter to solve this issue?
Thanks