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I am completely a noob to biology and DNAs, but recently I am looking into EM-algorithm used in the case of re-constructing haplotypes from genotype data.

I am just wondering what exactly in the process of DNA data collection prevents us from observing haplotypes directly.

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I am not completely sure what you mean. In DNA extraction, not even chromosomes are (usually, it is sometimes possible) singled, much less alleles. You have a complete bunch of chromosomes that you shear, most of the time put through PCR, tag with adapters and sequence. Often you actually even sequence several different probes together, so you additionally add a barcode to sort them afterwards. There are sequencing errors and different alleles and you have to figure out which read belongs where ... but this would be an answer, not a comment. Is this what you want to know more about? –  skymninge Jul 19 '13 at 7:00
    
To fix the problems that @skymninge pointed out EM (Expectation maximization) algorithms are used which try to find the likelihood of a read arising from a location –  WYSIWYG Jul 19 '13 at 11:00

2 Answers 2

The difficulty of reading haplotypes 'directly' lies in the difficulty of reading any long sequence of DNA, and this is for technical reasons.

Short DNA fragments are sequenced using a variety of methods. Gel electrophoresis can pull these fragments apart by length, but all the fragments have to be relatively close in size to get the accuracy needed to tell fragments that differ by one base pair apart. It's easy to separate a 16-bp segment from a 17bp segment easily, but it's much harder with a 516bp segment and a 517bp segment. Modern sequencing can sequence reads up to about 5kbp in length at a time. The 'reads' are then reassembled.

There are next-generation methods(pyrosequencing, etc) that can read a long dna molecule one base at a time, but for practical reasons sequencing an entire chromosome this way is cost and time prohibitive. As a result, there are always going to be fragments that you need to reassemble computationally, no matter how the sequencing is done.

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Are you asking why it is difficult to tell whether a cell is homozygous or heterozygous? If so, this is usually a combination of sensitivity of the assay (rtPCR can be optimized for this purpose - at least, I've heard of people doing it). Another aspect of this is that even if the alleles code for different things (eye pigment or something), much of the gene may be similar enough that PCR primers are difficult to construct in a way that they distinguish between the two alleles.

Then again, sometimes you get lucky and there's an EcoRI site right in the middle of one allele, but not the other.

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