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I need to prove that a given region on the genome maintains has the same base composition proportions (20% A, 15% T, 19% C, etc…) of that genome. I thought about doing Chi-squared test of independence since I deal with exact counts. As a result, I found $p<0.05$ suggests that they are independent so I say the base composition was changed. I just wanted to get a review of my action.

mydf:
                hg19      AR
            A 854963149  705448
            T 856055361  705519
            G 593325228  482498
            C 592966724  479646
            N 239850802 2806685

chisq.test(mydf)

Pearson's Chi-squared test

data: mydf X-squared = 15718000, df = 4, p-value < 2.2e-16

Is this method valid ?

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    $\begingroup$ Look at the data. Why are there so many Ns (presumably unknowns) particularly in the AR region? The actual genome is all TCGA, and Ns represent errors or technical difficulties in getting the correct sequence. Fix the data before you worry about how to analyze them. $\endgroup$ – EdM Mar 19 '17 at 12:57
  • $\begingroup$ @EdM I did not create this data. Therefore, I dont think I can do anything with those Ns unfortunately. I used published reference genome hg19 based on genome.ucsc.edu/cgi-bin/hgGateway?db=hg19 $\endgroup$ – MorTunco Mar 19 '17 at 18:37
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Technically what you have done is fine; a chi-square test is appropriate for this type of count data in defined categories.

The problem is interpreting the result. N represents an unknown base in the genome data alignment, not a specific base (A,T,C, or G). Over the whole hg19 human genome of 3.1 billion bases there are only 7.6% N (unknown) base calls, but your AR region has over 50% N, the major contribution to your highly significant chi-square statistic. AR must be a very hard to sequence region.

I certainly wouldn't include N calls in an estimate of "base composition," which biologically should only consider ATCG calls. It's impossible to know what the true A, T, C, G values are for over half of the AR region; the unknown positions might, say, be biased toward high C/G composition. So I don't know that you can come to any reliable conclusion about the "base composition" of region AR except that it's much harder to sequence reliably than most of the human genome.

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  • $\begingroup$ I did not realise the fact that huge percentage of my bases are Ns in ARs. Thank you for pointing this out and spending time answering my question. $\endgroup$ – MorTunco Mar 20 '17 at 6:52

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