FAQ & Definitions
Chance, Probability & Expectation Values
E-valuesMy (oversimplified) understanding of E-values is that they are a measure of the probability of finding a better alignment by chance (using random query sequences of the same length). The smaller the E-value the more confidence you can have in the likelihood the alignment is "real". For a detailed explanation, see The Statistics of Sequence Similarity Scores.
Quality values (QV)A quality value is formally QV = -10•log10(Pe)
Where Pe is the probability of error. Base calling programs use empirical data (known sequences), peak shape, spacing, resolution, type of terminator, gel matrix and other parameters to estimate Pe.
Typical high quality unmixed bases will have QV from 20 to 50.
Typical high quality with mixed bases (SNPs) will have QV from 10 to 50.
For a table of QV, Pe and more info on this important parameter read Phred_quality_score.
Clear range length
The clear range is the length of read after trimming the 5' and 3' ends so fewer than 4 bases out of 20 have a QV of less than 20.
The sample score is defined as the average quality value of the base calls within the clear range.
How do I sequence a PCR product directly (i.e. without cloning it)?
- PCR amplify the region of interest from your purified genomic DNA. Ideally, your PCR primers should be at least 50 nts from the region of interest. Call me if this is not the case.
- Check the product on an agarose gel (1.5% metaphor or other high quality agarose) or an appropriate non-denaturing PAGE gel.
- Purify the PCR product:
- a. IF (2) shows you have a single product, then use a simple PCR cleanup kit (Qiagen, Promega, Sigma, others) to remove primers and unincorporated dNTPs. Go to 4a.
- b. BUT IF (2) shows more than one product from your PCR reaction, then use use the simple cleanup kit and go to 4b. OR make the PCR conditions more stringent until you get a single, unique product. Then clean it up (3a) and sequence as in 4a OR try PCR Rescue: Making one band from many The bitesizebio.com PCR-Rescue method for gel separation of multiple products, capture the fragment of interest, and reamplify.
- Sequence the purified PCR product
- a. directly with one (and in a separate sample use the other from the opposite direction) of the PCR primers.
- b. Use a nested (aka internal) sequencing primer with the purified PCR product. This, in theory, should only bind to the region of interest.