Very low mapping rate of a bam file, reasons?

Question

[nidoo.beg]

I have got following results for my bam file.

186895644 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
1627664 + 0 mapped (0.87%:-nan%)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (-nan%:-nan%)
0 + 0 with itself and mate mapped
0 + 0 singletons (-nan%:-nan%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

How to interpret these values? And is this a good alignment for bam file to proceed further?

Thanks in advance!

[James Ashmore]

It's hard to give advice without knowing what your experiment is, but the flagstat shows that you tried to align 186895644 reads, but only 1627664 reads aligned (0.87%). This is an extremely low mapping rate, but without knowing what you are assaying here, you need to decide if this is appropriate.

[nidoo.beg]

iam aligning loxl1 gene obtained for exfoliation glaucoma patients(retina cells) on chromosome 15 to find SNP,s (single nucleotide polymorphism).Loxl1 gene is present on chromosome 15 so iam interested in aligning it on chr15..

[nidoo.beg]

iam aligning loxl1 gene obtained for exfoliation glaucoma patients(retina cells) on chromosome 15 to find SNP,s (single nucleotide polymorphism).Loxl1 gene is present on chromosome 15 so iam interested in aligning it on chr15..

[nidoo.beg]

iam aligning loxl1 gene obtained for exfoliation glaucoma patients(retina cells) on chromosome 15 to find SNP,s (single nucleotide polymorphism).Loxl1 gene is present on chromosome 15 so iam interested in aligning it on chr15..

[Antonio R. Franco]

what sequencing technology did you use ?

[nidoo.beg]

these reads are of loxl1 gene obtained from illumina.

[Antonio R. Franco]

Illumina is fine. You have mapped to Chr15 only I will try to map to the whole genome, to see what happens

[Rohit]

Reference you used might be wrong, also the mapped ones could be the most common genic regions between various species, just rRNA regions too

[nidoo.beg]

iam using chromosome 15 as a reference as i have NGS data for loxl1 gene responsible for exfoliation glaucoma and i want to identify SNP,s..

[GenoMax]

When you ask questions on Biostars this kind of information is critical and should have been provided in the original question.
Still 0.87% alignment seems low. Is your data from targeted/capture sequencing?

[nidoo.beg]

its targeted genome sequencing, i,ll repeat steps again and will see the results. thanks for responding..

[GenoMax]

You need to specify the size of the targeted region (was it just that one gene)? It kind of seems an overkill to use NGS for one gene. What does rest of 99% of the file represent?

[nidoo.beg]

yes it is just one gene obtained from 50 cases(patients)..

[nidoo.beg]

Then what is the easiest way to detect SNP,s in just one gene without over-killing NGS??

[Devon Ryan]

Sanger sequencing might be cheaper.

Answer 1

[Devon Ryan]

Less than 1% of your reads had alignments. That would normally indicate that the results are useless and that something went wrong.

Answer 2

[Sukhi Singh]

Very low mapping rate , (1627664/186895644)*100 = 0.87%

Was the correct genome assembly used? Run the file via fastQScreen to rule out cross-contamination.

[nidoo.beg]

yes i had done this step. thanks alot

Answer 3

[agata88]

You have total QC (quality control) passed reads : 186895644 but mapped only 1627664 (0.87%). Do you have PE reads? Because none of the reads were property paired. In my opinion something went wrong, did you choose correct reference genome?

Best,

Agata

[nidoo.beg]

i think there is some issue with reference, i,ll do the steps again thanks

[nidoo.beg]

no . i have single end reads

Answer 4

[Antonio R. Franco]

What program did you use for mapping? What were its setting? What did you sequenced and what did you use as reference?