Depth of aligned fragments

Question

[saifulislam99121]

Hello Everyone,

I have done multiplex PCR of 13 human genes and then sequenced them. My research question was to find out the even amplification of all the fragments by multiplex PCR.

The sequencing data was cleaned, aligned with the human reference genome. I have the sorted BAM file. How may I know the depth of each fragment that is aligned? How may I answer my research question from this ?

Thank you for your help. I can provide my workflow if anyone needed.

Answer 1

[GenoMax]

How may I know the depth of each fragment that is aligned?

You mean how many fragments are aligned to each regions of interest? You can use either mosdepth (LINK) or pandepth (LINK) to get this information.

[saifulislam99121]

Thank you. I have tried mosdepth using the sorted bam file. The output is like this-

chrom   length  bases   mean    min max
chr1    248956422   9703    0.00    0   14
chr2    242193529   10048   0.00    0   13
chr3    198295559   2269420 0.01    0   12924
chr4    190214555   4698    0.00    0   13
chr5    181538259   7815    0.00    0   10
chr6    170805979   4612    0.00    0   7
chr7    159345973   3147342 0.02    0   22213
chr8    145138636   8438    0.00    0   12
chr9    138394717   6588    0.00    0   71
chr10   133797422   14156   0.00    0   53
chr11   135086622   9623    0.00    0   22
chr12   133275309   4346    0.00    0   11
chr13   114364328   12717   0.00    0   252
chr14   107043718   1691    0.00    0   4
chr15   101991189   3704    0.00    0   23
chr16   90338345    2490770 0.03    0   22651
chr17   83257441    5344043 0.06    0   19631
chr18   80373285    3201    0.00    0   6
chr19   58617616    8262    0.00    0   20
chr20   64444167    5911862 0.09    0   37927
chr21   46709983    1023    0.00    0   2
chr22   50818468    3169    0.00    0   6
chrX    156040895   1717131 0.01    0   11298
chr1_KI270706v1_random  175055  61  0.00    0   1
chr1_KI270712v1_random  176043  193 0.00    0   2
chr14_GL000009v2_random 201709  201 0.00    0   2
chr14_GL000225v1_random 211173  244 0.00    0   2
chr22_KI270735v1_random 42811   64  0.00    0   1
chrUn_KI270743v1    210658  201 0.00    0   2
total   3032059866  20995326    0.01    0   37927

How may I understand the read number or depth from this?

Thank you for your help. Please let me know if I miss something.

[GenoMax]

You can provide a BED file here but looks like you already have the result from pandepth below. Here chr3 shows a mean coverage of (12924). This is slightly different from pandepth result (12098) but similar. Probably because of differences in how reads are calculated in two programs.

[saifulislam99121]

I have also used pandepth. But I could not understand the results. How can I calculate the read number? I have attached a picture of the output. I have used both bam and bed file in this case.

[GenoMax]

Header row is self explanatory. It tells you how many reads there are covering the region (TotalDepth) and the mean depth in last column.