Week 11 of SIP

Hi everyone!! =) I'm back after 4 weeks. 11 weeks has passed and 9 more weeks attachment will come to an end.

I'm basically doing the same things everyday and week since I'm not attached to a routine lab. So it's just PCR, purification, sequencing, blasting, genotyping and data entering. And this repeats.

In my previous post, I discussed about what blasting is and what was done. This post, i will talk about genotyping.

Genotyping
This step is after blasting. After all the SNPs are found, you have to genotype all the samples for each and every SNP. You have to screen and look at all the samples and find the SNP. Before you can find the SNP, you have to copy a small fragment of the sequence either in front or behind that particular SNP from the reference sequence of a gene. Usually, the fragment is around 8-10 base pair long. Noone will copy a super long fragment to find in the DNA sequence of the samples. Similarly, too short the fragment won't be copied as well. This is because if the fragment is too short (around 4 or 5)- eg:AGTC, you may not be able to find that particular SNP. Possible reason why is 'cause the sequence 'AGTC' may be found at more than one postion on the DNA sequence. That is, for example at position 1103 there's 'AGTC' and at position 330, there's another 'ATGC'. Thus, it's important not to copy too short a fragment to find on you samples' DNA sequence.

After copying the suitable length of fragment in front or behind the SNP, you can start screening every samples. For example, the SNP is 334A>G. You have to screen every samples to see at the 334th position whether the subject has AA or AG or GG. In this case, AA is known as the wild type, AG is known as the heterozygous variant while GG is known as the homozygous variant.

Depending on the number of SNPs you have found during blasting and also the number of samples you have, you will have to screen that many times. For example, you have 100 samples and found 87 SNPs, you have to screen a total of 8700 times. It's a quite tedious process but it's necessary.

Genotyping is done using the programme Sequencing Analysis and similar to blasting, you have to look at the computer screen again.

After all the SNPs are genotyped for all the samples, you have to enter them into Microsoft Excel; data entering. In my company, it's programmed in such a way that we do not have to calculate the genotypic and allelic frequency.

Genotypic frequency is the frequency of a certain genotype in a population. For example, the frequency of Indians having AA for the SNP 334A>G. As for allelic frequency,it is the frequency of the population getting a paricular allele (eg: A) of a SNP (eg:334A>G) in their genes.

Allelic freqeuncy is expressed in percentage and it is calculated by taking the number of people in the population having a paricular genotype (AA) and multiply by 2 ('cause there's 2 A in the genotype AA). After that, plus the number of individuals having AG. For this value, you do not have to multiply by 2 'cause AG has only one A. After getting the value, divide it by the total number of alleles.

Example:
Number of people having AA= 5
Number of people having Aa= 4
So, take 5X2 and then add 4 to the vaule.
Total number of people= 10
So, total number of alleles= 10x2= 20
Thus, the allelic frequency for A in this paticulat population is 14/20= 0.7, which is also 70% of the population.

Genotypic frequency is easier to calculate. It is just taking the number of people having AA and divide with the total number of people in the population. Genotypic frequency is also expressed in percentage.

Example:
Genptypic freqency getting AA= 5/20= 0.25= 25%

After getting the allelic and genotypic frequencies, you will have to see if they are in Hardy-Weinberg equillibrium (HWE). I will touch on this in the next post; the principle of HWE, how to calculate and etc.

Hope my post is easy for you guys to understand. =)

Take cares everyone. See you guys at the next coming campus discussion.

Lyn
0611027D
TG02