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04032012, 09:12 AM  #1 
Junior Member
Location: Boston, MA Join Date: Apr 2012
Posts: 2

How do I calculate the probability of sequence coverage at particular window?
Hi all,
A newbie question: How do I calculate the probability of a random set of sequences (at a specified length, say short reads of 25bp) aligning to a set window length (say 10kb)? Essentially, I'd like to know the sequence coverage probability along a specified length of DNA. I'd like to use this sequence coverage probability to test whether what I see (for example, say I see 3 reads within a particular 10kb window) is truly significant or aligned by random chance. Please let me know your thoughts and whether this is a valid question to ask in the first place. Thanks! 
04032012, 11:12 AM  #2 
Senior Member
Location: Heidelberg, Germany Join Date: Feb 2010
Posts: 994

If I understand your question correctly, you want to calculate the probability that a read with a completely random sequence aligns to a place in your genome by chance. The answer is usually: zero.
There are 4^25=1.1e15 possible 25bp reads. The human genome has 3e10 base pairs, hence the probability of a random 25mer actually occurring in the human genome is roughly 3e10/1e15=2e6. Hence, if you see a read aligning somewhere, it has almost certainly been amplified from a real biological template, i.e., it is either from the sample or from contamination. Contrary to popular belief, there is no such thing as alignment noise in highthroughput sequencing. Last edited by Simon Anders; 04032012 at 11:13 AM. Reason: corrected distorting grammer mistake 
10222013, 03:58 AM  #3 
Member
Location: UK Join Date: Sep 2012
Posts: 61

Hello guys
I am having almost the same problem, and i am confused of how to calculate the probability, here is my issue: I have sequenced a PCR fragment of 2kb from original reference sequence of 7.5kb. I used illumina HiSeq pairedends technology to generate 5 million 80bp reads with a coverage of x30 as I am looking for a recombination event between two serotypes of viruses, which is rare event. I know that the event occurs by 1%, so I expect to find 1% of the reads represent the recombination event. Among these reads there are some reads which are going to span the junction point of the Recombinants, and therefore not aligned to any of the reference sequences. I want to calculate the probability of the coverage of those reads which span the junction point to calculate the error rate between the expected and the observed. Need help! Many thanks 
10222013, 04:13 AM  #4 
Member
Location: UK Join Date: Sep 2012
Posts: 61

Hi again
I forgot to add that the junction point could be occur at any nucleotide lies in the 2kb fragment. Thanks a lot 
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