We have reviewed from start to finish the next generation sequencing wet bench process, data review and troubleshooting. I’d like to take a more in-depth look at the types of variants that can be detected by the targeted amplicon NGS panels that our lab performs: single nucleotide variants, multi-allelic variants, multi-nucleotide variants, insertions (including duplications), deletions and complex indels. In our lab, we review every significant variant and variant of unknown significance in IGV to confirm the call is made correctly in the variant caller due to the difficult nature of some of these variants. I have included screenshots of the IGV windows of each of these types of variants, to show what we see when we review.
Single Nucleotide Variants (SNV)
The most common (and straight forward) type of variant is a single nucleotide variant – one base pair is changed to another, such as KRAS c.35G>A, p.G12D (shown below in reverse):
A multi-allelic variant has more than one change as a single base pair (see below – NRAS c.35G>A, p.G12D, and c.35G>C, p.G12A – shown below in reverse). This may be the rarest type of variant – in our lab, we have maybe seen this type in only a handful of cases over the last four years. This could be an indication of several clones, or different variants occurring over a period of time.
Multi-nucleotide Variants (MNV)
Multi-nucleotide variants are variants that include more than one nucleotide at a time and are adjacent. A common example is BRAF p.V600K (see below – in reverse) that can occur in melanoma. Two adjacent nucleotides are changed in the same allele. These variants demonstrate one advantage NGS has over dideoxy (Sanger) sequencing. In dideoxy sequencing, we can see the two base pair change, but we cannot be certain they are occurring on the same allele. This is an important distinction because if they occurred on the same allele, they probably occurred at the same time, whereas, if they are on different alleles, they were probably two separate events. It is important to know for nomenclature as well – if they are on the same allele, it is listed as one event, as shown below (c.1798_1799delGTinsAA, p.V600K) as opposed to two separate mutations (c.1798G>A, p.V600M and c.1799T>A, p.V600E). As you can see in the IGV window below, both happen on one strand.
Insertions are an addition of nucleotides to the original sequence. Duplications are a specific type of insertion where a region of the gene is copied and inserted right after the original copy. These can be in-frame or frameshift. If they are a replicate of three base pairs, the insertion will move the original sequence down, but the amino acids downstream will not be affected, so the frame stays the same. If they are not a replicate of three base pairs, the frame will be changed, causing all of the downstream amino acids to be changed, so it causes a frameshift. A common example of a frameshift insertion is the 4bp insertion in NPM1 (c.863_864insCTTG, p.W288fs) that occurs in AML. In IGV, these are displayed by a purple hash that will show the sequence when you hover over it.
Deletions, on the other hand, are when base pairs are deleted from the sequence. These can be in-frame or frameshift, as well. An example is the 52bp deletion (c.1099_1150del, p. L367fs) found in the CALR gene in cases of primary myelofibrosis or essential thrombocythemia.
Lastly, NGS can detect complex indels. These, again, are a type of variant that we could not distinguish for sure using dideoxy sequencing. We would be able to detect the changes, but not whether or not they were occurring on the same strand, indicating the changes occurred at the same time. The first example is a deletion followed by a single nucleotide change – since these both occur on the same strand, they most likely occurred together, so they are called one complex deletion/insertion event (KIT c. 1253_1256delACGAinsC, p. Y418_D419delinsS). First the ACGA was deleted, then a C was inserted.
The last example involves multiple nucleotides changes all in the same vicinity (IGV is in reverse for this specimen as well). Using HGVS nomenclature as in all the previous examples, this would be named RUNX1 c.327_332delCAAGACinsTGGGGT, p.K110_T111delinsGV.
-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine.