BLAST

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Chapter 8: 20 Tips to Improve Your BLAST Searches

8.12 Use Caution When Searching Raw

Sequencing Reads

The largest source of raw sequencing reads comes from the early stages of genome

projects and from EST sequencing. Most sequencing reads have an error rate of

about 1 percent. This rate isn’t uniform; there is a spike near the beginning and a

gradual increase towards the end of the read. In addition, some regions have intrinsi-

cally high error rates due to compositional properties such as high GC content. DNA

sequencing involves several steps, and there are abundant opportunities for mechani-

cal and human error. Thus, you will need to be careful when using large word sizes.

For redundant sequence collections, such as 3x shotgun coverage of a genome, large

word sizes are fine, but if the absence of a single alignment is troublesome, scale

down the word size to keep sequencing errors from preventing seeding.

Raw sequencing reads may be contaminated from a variety of sources. Cloning vec-

tors are one expected source. Depending on the sequencing center, the vectors may

or may not have been clipped from the sequence. Other kinds of contamination are

also possible. Nuclear DNA is sometimes contaminated with mitochondrial or viral

DNA, and any collection of sequence can be contaminated ...