SeqStudio Flex Genetic Analyzers

Amplification of DNA sequences by polymerase chain reaction (PCR) remains the workhorse for almost all aspects of molecular biology and genomic research.  There is an increasing need to analyze many targets in a single sample.  However, this can be tedious by PCR, involving setting up individual reactions for each of the desired targets.  Methods that can analyze multiple sequences from a single sample would simplify workflows and conserve precious samples.  One method to address this is fragment analysis by capillary electrophoresis (CE).  CE separates amplicons based on both size and fluor in a single capillary allowing a very large number of targets to be analyzed within a single sample.  

Multiplexed PCR coupled with fragment analysis presents a tremendous opportunity to perform complex analyses with minimal expense or effort.  Experiments are limited only by the ability to design primer sets against the targets of interest.  It can therefore be adapted and utilized in nearly any scenario where detection of many target sequences in a single sample is desired.  

This example demonstrates the power of multiplexed PCR by fragment analysis using a custom panel for detecting respiratory viral pathogens.  A panel of PCR primers containing 6-FAM labeling at the 5’-end were designed to detect 12 different respiratory RNA viruses so that each organism generated a different sized amplicon.  The resulting amplicons could be separated and examined using the SeqStudio, SeqStudio 24 Flex, or 3500xL genetic analyzers.

It is often necessary to run a quality check (QC) on a starting pool of double-stranded DNA (dsDNA) before initiating genomic analysis or continuing to the next step. For example, DNA extracted from FFPE-preserved tissue may be fragmented and too small to provide useful information, so it’s useful to know the overall size before proceeding.

The size and relative abundance of dsDNA fragments can be analyzed on the SeqStudio Flex Series genetic analyzers. In the protocol, TOTO-1, a fluorescent DNA intercalating dye, is incubated with a sample, which is run through POP7 polymer with the capillary heater turned off. (This maintains the DNA in a largely double-stranded, non-denatured state.) By comparing the resulting migration of the test fragments with the migration of known dsDNA standards, the size of the test fragments can be determined.

In this test, we assessed the performance of the SeqStudio Flex Genetic Analyzer as well as the 3500xL Genetic Analyzer in analyzing several kinds of DNA samples, including two known dsDNA standards shown in the figure. Panel A illustrates an analysis of phage lambda DNA digested with HindIII, showing the familiar pattern of eight fragments ranging from 23,130 to 125 bp. Panel B shows a mixture of the same lambda HindIII digests with phage phiX174 digested with BsuRI. In addition to the eight lambda fragments, ten more fragments ranging from 1,353 to 72 bp were detected. The profiles obtained on the SeqStudio Flex (left) and 3500xL (right) instruments were quite similar, validating the newer instrument versus the 3500 series.

Some investigations require focused sequence information for a specific region of a genome. While genome-wide discovery tools such as NGS provide vast amounts of data over very large regions, such complex approaches are not always needed in focused research. Sanger sequencing provides a simple, cost-effective, and easy-to-interpret solution for gold-standard sequencing of a genome region. 

To demonstrate focused sequencing, we sequenced the region of the SARS-CoV-2 genome that encompasses the Spike protein and adjacent sequences, relying on previous research identifying 12 pairs of sequencing primers for the entire SARS-CoV-2 Spike gene. The Spike gene region of SARS-CoV-2 covered by the 12 overlapping amplicons extends across 5 kb.

In panel A of the figure, high-quality Sanger sequencing results (indicated by the height of the light blue bars) were obtained from nearly all 12 amplicons in both directions as shown in the figure.

These results were assembled into a single contig read of the SARS-CoV-2 Spike gene. The read quality metrics were nearly identical on all three analyzers. Consistent base-calling quality, as demonstrated here, enables Sanger sequencing to assemble the sequence of focused regions using overlapping mid-length amplicons, generating contigs that cover a locus of interest.