Sanger sequencing for a single amplicon

A single cleaned amplicon is a Sanger question. One template, one sequencing primer, one chromatogram. The decision this page supports is whether that experiment is the right witness for the sequence you want to write down, or whether you have already wandered into a mixture that a capillary cannot adjudicate. The surrounding path from libraries to many reads at once is next-generation sequencing from library to reads. Sanger stays the direct reading of one molecule population that you have already reduced to one band.

People reach for it after cloning a plasmid, after amplifying one exon, or after a genome edit when they want the alleles in a colony as peaks rather than as a model. It is a poor way to survey a genome and a poor way to see a variant present in only a small fraction of molecules. Reagent classes for the bench sit in the genomics and sequencing catalogue. The design of the study, if the amplicon is one piece of a larger project, belongs with the genomics research pathway.

Chain termination writes length as a colour

Sanger sequencing is chain termination. A polymerase copies the template from a primer. Most of the building blocks are ordinary deoxynucleotides. A small fraction are dideoxynucleotides, which lack the hydroxyl that would let the chain grow. When the polymerase incorporates a terminator, that molecule stops. Across millions of copies, stops happen at every position. The fragments differ in length by single bases.

In the fluorescent form used on modern capillaries, each terminator carries a different dye. A separation sorts the fragments by length, shortest first. A detector reads the dye colour as the fragments pass. The chromatogram is that time series: one peak per incorporated terminator, four colours for the four bases. The base at position 40 is the colour of the fragment that stopped at length 40. Nothing in that picture counts independent genomes the way a deep pileup does. You are looking at the ensemble average of one priming event on one template preparation.

Usable base calls from that ensemble are often under one kilobase even when the amplicon is longer. Peaks are sharp near the start of the useful window and broaden as fragments get long, because separation loses resolution. Plan a second primer further along if the amplicon exceeds what one read can cross. Do not treat a long, noisy tail as extra evidence.

What has to be in the tube

The reaction class is a polymerase, a buffer the enzyme's instructions specify, the four deoxynucleotides, the four dye terminators, one primer, and the template. Kits bundle those. Follow the amounts and the cycling programme on the insert that matches the kit in your hand. A table copied from a different enzyme will change the balance between extension and termination, and the peaks will either vanish or pile up.

The template is either a cleaned PCR product or a plasmid. Leftover PCR primers will prime as well, which is a second sit-down you did not order. Leftover nucleotides change the terminator ratio. Cleanup is its own class of method: an enzymatic chew of primers and nucleotides, a column, or beads. Pick one and confirm, on a gel or a size trace, that the product is still one band afterwards. Gel conditions that make a doublet visible are in agarose gel electrophoresis for DNA.

The sequencing primer is not automatically one of the PCR primers, though it can be. It must anneal at one place on this molecule. Check that site against the sequence you believe you amplified, with a tool such as NCBI Primer-BLAST when the template is a genome, or by a direct search when the template is a plasmid map. A primer that sits down twice produces two ladders in one capillary. The trace looks like noise from the first peak, and no basecaller will rescue it into a single sequence.

Read the trace, then choose a branch

Start from the chromatogram, not from the letters a program printed underneath it. The letters are a convenience. The peaks are the data.

The first stretch often contains dye blobs: broad, humped signals from unincorporated terminators that survived a weak cleanup. They are not bases. Begin calling where the peaks become narrow and evenly stepped. If blobs swallow the region you care about, clean the reaction again. Adding more template on top of a dirty reaction usually reprints the blob.

Then walk. Clean, single-colour peaks that match the spacing support a base. A double peak at one coordinate, with both colours strong and the neighbours clean, supports two bases at that position in the molecules that were primed. In a diploid PCR that is the usual heterozygous pattern. In a plasmid prep it means two plasmids in the well, or a primer problem. Those are different biological stories. The trace does not say which until you know what you loaded.

If the trace is clean and then becomes two overlapping sequences that never resolve, an indel is the usual explanation. The polymerase read both alleles in register until the insertion or deletion, then the lengths diverged. You cannot call the downstream sequence from that mixture. Clone the amplicon and sequence several colonies, or design an allele-specific approach. The companion note on checking whether a genome edit worked walks through that split in an editing context.

Branch when the whole trace is weak. A weak trace is low signal, not a mysterious genotype. Check template amount against the kit's accepted range, check that the primer matches this construct, and check that the amplicon was actually in the tube. A no-template control that produces a readable trace means the product is a contaminant. Stop and return to PCR controls and contamination control before you edit any reference sequence.

Failure modes that look like a genotype

A compression is a local mobility artefact. Peaks squash together, often in GC-rich stretches, and the basecaller may insert or skip a base. The reverse read, which crosses the same stretch from the other direction, usually does not compress in the same place. That is why one direction is a sketch and two directions are the record.

A slipped primer, or a primer with a mismatch near its 3-prime end, can still extend and then tell you the sequence of the wrong site with beautiful peaks. Identity of the amplicon is a gel-size hint plus a match to the reference you named, on a build you named. Ensembl is one public place to keep that reference honest. A perfect-looking trace of the wrong exon is a successful reaction and a failed experiment.

Heterozygous indels and two-colony plasmid preps are the mixtures people try to read through. Do not. Mixed peaks mean a mixture. Average them in your head and you will invent a protein sequence that no molecule has.

Dye blobs at the start are chemistry, not heterozygosity. They are wide, they often involve several colours at once, and they sit where the shortest fragments run. If a claimed variant lives in the first few dozen bases, move the sequencing primer back so the variant falls in the resolved window, or read it from the other end.

None of this is a variant caller. There is no depth in the coverage sense. A strong peak is a strong average of the molecules that took this primer, not one hundred independent chromosomes. If the biological claim needs allele frequency, rare variants, or a structural change larger than this amplicon, the method class in how short reads and long reads differ is the conversation, and a single trace should not be asked to impersonate it.

The sample may still be infectious

A finished amplicon is nucleic acid, and the swab, culture, or tissue it came from may have been infectious. Whether extraction inactivated the organism, and which room may handle the lysate, is an institutional biosafety decision. This page does not make that decision and does not approve a diagnostic use. A research trace supports a research sequence. It does not become a clinical report because the peaks were clean. The practical frame for the bench is biosafety basics for research benches.

Heat, handoff, and a short run

A capillary run is short compared with a flow-cell run, and a power cut is less likely to bisect it. The fragile part is the handoff. Cleaned amplicons and primer aliquots left in a warm courier pouch, or on a windowsill while a building's cooling is down, are not the same templates you quantified in the morning. Humidity loosens adhesive on a dried sequencing plate. Write the holding temperature into the note that travels with the tube, and record the time the plate spent outside the cold. If the receiving bench is across the city, say so in the specification rather than hoping the peaks will confess the trip.

What to put in the enquiry

State that the job is one amplicon or one plasmid, the length you expect, the primer sequence and its annealing site, the cleanup you already did, and whether both strands are required. Mention the reference sequence the trace should be compared with, including the build if the template is genomic. Say what a mixed trace would mean for your decision, so a double peak is reported rather than forced into a single letter.

A single-amplicon trace can be discussed against the Sanger DNA sequencing enquiry reference. A question that has outgrown one primer pair can be discussed against the whole-genome sequencing enquiry reference. Send the specification with the quote request. The checks that belong in that specification before any instrument starts are in what to check before a sequencing run, and the habit of writing them down is in commissioning a sequencing or proteomics study.