Bioinformatics tools

What each tool is for

GenBank to FASTA strips a GenBank record down to the header and sequence that most alignment and BLAST tools expect. Reverse Complement gives the reverse, complement or reverse complement of a DNA sequence, which you need constantly when designing reverse primers or checking which strand a feature is on.

The Primer Tm Calculator estimates melting temperature with several methods, including the simple Wallace rule, a GC-based formula and nearest-neighbour thermodynamics with salt correction. The methods can disagree by several degrees on the same primer. Nearest-neighbour is generally the one to trust for PCR design; the Wallace rule is only a rough guide for short oligos. The GC Content Calculator reports base composition, and the FASTQ Quality Viewer plots per-position quality scores so you can decide where to trim reads.

A typical primer workflow

A common path through these tools: take a GenBank record from NCBI and convert it to FASTA, find the region you want to amplify, write the forward primer from the top strand and the reverse primer from the reverse complement of the far end, then check both primers. As a rule of thumb, aim for GC content around 40 to 60 percent and for the two primers' melting temperatures to be within a few degrees of each other, calculated with the same method.

Limits worth knowing

Because everything runs in the browser, very large files are limited by your device's memory. The FASTQ viewer is meant for a quick look at a sample of reads, not for whole sequencing runs; use FastQC or a similar tool on the command line for those. None of these tools replace checking a primer pair for secondary structure and dimers before you order it.