Biomedical Engineering Reference
In-Depth Information
Notes
z
Available from the website http://www.molgen.ua.ac.be/bioinfo/novosnp/. [22].
aa
For additional information, see the methods chapter in
Comparative Genomics
, volume 2 [23].
bb
novoSNP will run automatically and reads the trace files and performs base calling, aligns
reads to the reference sequence, and detects SNPs/indels. This can take a long time, depending
on the number of traces and the size of the reference sequence. Watch the program status bar
to keep track of the project.
cc
This opens up a new window with all read alignments to the reference sequence in which the
variant positions are highlighted.
dd
This opens a popup window with several automatic filtering options: sample name patterns,
which help NovoSNP identify samples by their trace names; base quality algorithm, which can
either be NovoSNP's own algorithm or Phred; and quality clipping, which removes low-quality
bases from the ends of reads.
ee
The default report type exports SNPs and indels detected by novoSNP into a tab-delimited file.
2.3 Troubleshooting
2.3.1 Primer design
Primer design typically fails for one of the following reasons:
•
Primer GC content
: In regions with unusually high or low G
+
C content, primer design
fails because the percentage G
C content falls outside the desirable range (20 - 50%), or
because potential primers have melting temperatures that fall outside the permitted range.
Try adjusting the permitted maximum melting temperature first and then the allowable
percentage G
+
+
C.
•
Repetitive sequence
: Often a region of interest is flanked by repetitive sequence such
that unique primers cannot be designed on both sides. Such regions are typically resistant
to most primer-based assays. However, occasionally PCR will succeed if one (and only
one) primer is designed within a repetitive region.
•
PCR product size
: If candidates are available for both forward and reverse primers, but
no suitable assay can be designed, try adjusting the PCR product size from 80 - 400 bp to
80 - 1000 bp.
2.3.2 PCR amplification
A number of variables can contribute to PCR failure. Some of the most common issues are:
•
Variable primer annealing sites (SNP-in-Primer)
: When PCR primers are designed
across a sequence variant such as an SNP, PCR failure or allele dropout (loss of a single
allele during PCR) may occur, particularly if the variant is near the 3
end of the primer. To
avoid such issues, use an SNP-marked reference sequence when designing PCR primers.
Variable positions marked with Ns will not be considered for primer design.
