This can be problematic for SNP calling, although the higher average coverage is advantageous. Exome sequencing projects usually have much higher average coverage, but the current technologies have biases that create very uneven coverage over the exome. However, coverage is usually quite uniform, with about the same number of reads at each location so that the quality of SNP calls is even over the genome. in family studies we could consider the genotype of relatives at that location.īecause it is so expensive, coverage for whole genome sequencing is generally low. However, if there is 1 A and 5 T's, we would assume that the A is a sequence error unless we have information from other samples that suggest that it could be correct - e.g. So if a location is covered by 6 reads and there are 2 A's and 4 T's we would consider the individual to be a heterozygote at that locus with 2 alleles, A and T. Typically, when calling a SNP at a location, we should have at least 2 reads with each variant. However, high throughput sequencing is getting more accurate all the time. The SNP call error rate is about 3% with Sanger sequencing (which is better than high throughput). To account for the various types of error in the data, we only call new SNPs at locations that have multiple reads (usually at least 75 for de novo calls) and if either the vast majority of the reads match each other but not the reference at this site, or if two variants are in about 50/50 ratio. Finally, alignment problems can cause errors, if a closely related sequence is wrongly mapped to the site. We also have to acknowledge that the reference itself might have an error at some locations (although for the human genome this is less problematic than for other less well-researched genomes). Usually we assume that exact replicates are technical errors and remove all but one. SNPs in the human genome are about bp and about 1-2% of reads are exact duplicates which may be due to technical issues in sample preparation so that seeing two reads with the same variant is not sufficient to conclude that a SNP is present. They are about the same frequency as some of the SNPs that we are interested in detecting. One problem is that the best sequencing methods still have error rates of a couple of percentage points. In the case that the individual matches the reference, we only say that a SNP is present at this location if we have determined in advance that there is genetic variation in the population at the location. If all the reads have an A or a T at the SNP location, the individual is a homozygote. However, after the reads are aligned you can detect single nucleotide mismatches. Here's an example of reference and mapped reads:Īs you can see in this reference above these SNPs are not necessarily in the middle of the read. The fragments are sequenced and then mapped to the reference without the requirement of perfect match at each location. SNP Detection with a Reference GenomeĪs with other high throughput sequencing analyses, the workflow begins with fragmented DNA from a tissue. However, in this section, we will briefly go over SNP calling using sequencing. We are going to focus on finding the association of SNPs with phenotype. Finally, SNP analysis is now being used for breeding of domesticated plants and animals to improve yields, nutritional value etc. Other uses of SNP analysis is for inferring evolutionary relationships. The genotype might be causative or it might associated with something else that is actually associated with disease - for example, a genotype associated with obesity might be due to its association with a phenotype such as a glucose processing phenotype. In human populations, we are often interested in disease phenotypes and the associated genotypes. As well, resequencing parents and off-spring allow researchers to determine how frequently new variants arise in a population. Resequencing projects allow researchers to determine the extent of genetic variation in a population. In a single genome, genomic variation can be determined only at sites at which the individual is heterozygotic. This type of project is called "resequencing" because the initial sequence can be used as a reference making the analysis of each additional individual's DNA much simpler. While the initial draft of the human genome was published in 2000, there have been many subsequent efforts to add to our knowledge of the human genome by assembling genomes for many different individuals.
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