BLAST

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Chapter 8: 20 Tips to Improve Your BLAST Searches

8.17 Perform Pilot Experiments

Before embarking on a large BLAST experiment, first try some pilot experiments. For

example, if you want to compare all human proteins to all nonhuman proteins, try

100 proteins first. Or, if you want to annotate a 5 mb chromosomal region with

BLASTX similarities, search 100 Kb first. If you’re unsure of which parameters to

use, try several and see which ones give you the kinds of results you’re looking for. It

may seem like a waste of time, but performing pilot experiments will actually save

you time in the end.

8.18 Examine Statistical Outliers

In a high-throughput setting, BLAST reports may be huge and number in the thou-

sands. There’s no way you can look at all of them, but for quality control, you

should examine some of them. Keep global statistics on BLAST reports, such as

number of hits per Kb. Statistical outliers may point to general problems that

become more apparent in certain sequences.

8.19 Use links and topcomboN to Make Sense of

Alignment Groups

WU-BLAST has two very useful parameters for displaying alignment groupings.

topcomboN sorts alignments into groups and labels them. The links parameter shows

the order of alignments in a group, which is much like the order of a gene’s exons.

Figure 8-9 displays these features.

8.20 How to Lie with BLAST Statistics

Several techniques can help you massage BLAST statistics to either hide significant

alignments or make meaningless alignments appear highly significant. Why would

you want to do this? If you have to ask, you’re not the intended audience. Dishonest

evil doers read on.

The easiest method to adjust the significance of all scores is to set the effective size of

the search space either higher or lower. Command-line parameters in both NCBI-

BLAST (

-Y) and WU-BLAST (Y and Z) are available. You can also alter the scoring

scheme by editing the scoring matrices. A more involved approach involves hacking

the source code to set your own values for λ, k, and H. WU-BLAST makes it all too

easy because you can alter scores or set Karlin-Altschul parameters on the command

line. Whatever approach you take, you will, of course, want to edit the footer to

cover your tracks. The easiest way to do this is to run the search twice and diff the

footers to determine what needs fixing.

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20 Tips to Improve Your BLAST Searches

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With low gap penalties, you can make alignments between just about anything. For

BLASTN, NCBI-BLAST always uses ungapped statistics, so you don’t have to do

much work to lie. Just hope that nobody notices all the gaps. This works best if you

have a supervisor who is either too busy to look at alignments or wouldn’t know a

decent alignment if it bit him. NCBI-BLAST is very restrictive about what gap penal-

ties you can employ for the protein-based BLAST programs. Your only choice here is

to hack and recompile. WU-BLAST is very easy; set your gap costs low and include

warnings on the command line to suppress messages about ungapped statistics.

Another way to trick the unobservant is to remove complexity filters. This works

especially well when claiming that some anonymous low-complexity region or tran-

script is a cool gene. You can almost always find a small ORF that has a poor match

to something with an interesting definition line. A poor match is only poor if you

don’t know how to fix the statistics. This approach even works when fooling scien-

tific journals. (It really does. We’ve seen it happen.)

Figure 8-9. WU-BLAST topcomboN and links (the top-to-bottom order of alignments in the

graphic (a) are the same as the statistics lines from the BLASTX report (b))

a

b

1

2

links topcomboN