• SNVs (single nucleotide variants), also known as single base substitutions, are the
simplest type of variation as they only involve the change of one base for another in a
DNA sequence. These can be subcategorized into transitions (Ti) and transversions
(Tv); the former are changes between two purines or between two pyrimidines, whereas
the latter involve a change from a purine to a pyrimidine or vice versa. An example of a
transition would be a G > A variant. If the SNV is common in a population (usually with
an allele frequency > 1%), then it is referred to as a SNP (single nucleotide polymorphism). A common post-calling analysis involves looking at the Ti/Tv ratio, which can
vary between 2 and 3 depending on the genomic region under analysis [14]. If this ratio
is far from the expected, it may indicate a large proportion of false positive calls.
• MNVs (multi-nucleotide variants), which are sequence variants that involve the consecutive change of two or more bases. An example would be one of the types of mutations
caused by UV irradiation, CC>TT. Similarly to SNVs, there are some MNVs that are
found at higher frequencies in the population, which are referred to as MNPs [15].
• Indels (portmanteau of insertions and deletions), which involve the gain or loss of one
or more bases in a sequence. Usually, what is referred to as indel tends to be only a few
bases in length. An example of a deletion would be CTGGT > C and an insertion would
be represented as T > TGGAT.
• Structural variants, which are genomic variations that involve larger segments of the
genome. These can involve inversions, which is when a certain sequence in the genome
gets reversed end to end, and copy number variants including amplifications, when a
fraction of genome gets duplicated one or more times, and larger deletions, when large
segments of the genome get lost. There is not a strict rule defining the number of base
pairs that make the difference between an indel and a structural variant, but usually, a
gain or loss of DNA would be called a structural variant if it involved more than one
kilobase of sequence.
Most variant callers identify SNVs, but there are only some variant callers that will
report indels or structural variation [3]. This is because usually the algorithms underlying
the detection of these types of variants tend to be quite different: SNV, MNV, and short
indel detection comprise the comparison of a pile of sequencing reads and their alignments
to the reference genome (as has been discussed throughout Chap. 9), whereas larger indels
and structural variant calling require calculating a distribution of insert sizes and detecting
those read pairs that fall outside it, as well as the direction of alignment of both mate pairs
[16].
It is also important to consider the type of sequencing that was used for the experiment.
For example, whole genome sequencing and whole exome sequencing have different
amounts of coverage, depth, and sequencing uniformity. Some variant callers such as
MuTect2 and Strelka2 show better performance in sequencing with higher average
sequencing depth and lower coverage [17].
10 Identification of Genetic Variants and de novo Mutations Based on NGS
131
simplest type of variation as they only involve the change of one base for another in a
DNA sequence. These can be subcategorized into transitions (Ti) and transversions
(Tv); the former are changes between two purines or between two pyrimidines, whereas
the latter involve a change from a purine to a pyrimidine or vice versa. An example of a
transition would be a G > A variant. If the SNV is common in a population (usually with
an allele frequency > 1%), then it is referred to as a SNP (single nucleotide polymorphism). A common post-calling analysis involves looking at the Ti/Tv ratio, which can
vary between 2 and 3 depending on the genomic region under analysis [14]. If this ratio
is far from the expected, it may indicate a large proportion of false positive calls.
• MNVs (multi-nucleotide variants), which are sequence variants that involve the consecutive change of two or more bases. An example would be one of the types of mutations
caused by UV irradiation, CC>TT. Similarly to SNVs, there are some MNVs that are
found at higher frequencies in the population, which are referred to as MNPs [15].
• Indels (portmanteau of insertions and deletions), which involve the gain or loss of one
or more bases in a sequence. Usually, what is referred to as indel tends to be only a few
bases in length. An example of a deletion would be CTGGT > C and an insertion would
be represented as T > TGGAT.
• Structural variants, which are genomic variations that involve larger segments of the
genome. These can involve inversions, which is when a certain sequence in the genome
gets reversed end to end, and copy number variants including amplifications, when a
fraction of genome gets duplicated one or more times, and larger deletions, when large
segments of the genome get lost. There is not a strict rule defining the number of base
pairs that make the difference between an indel and a structural variant, but usually, a
gain or loss of DNA would be called a structural variant if it involved more than one
kilobase of sequence.
Most variant callers identify SNVs, but there are only some variant callers that will
report indels or structural variation [3]. This is because usually the algorithms underlying
the detection of these types of variants tend to be quite different: SNV, MNV, and short
indel detection comprise the comparison of a pile of sequencing reads and their alignments
to the reference genome (as has been discussed throughout Chap. 9), whereas larger indels
and structural variant calling require calculating a distribution of insert sizes and detecting
those read pairs that fall outside it, as well as the direction of alignment of both mate pairs
[16].
It is also important to consider the type of sequencing that was used for the experiment.
For example, whole genome sequencing and whole exome sequencing have different
amounts of coverage, depth, and sequencing uniformity. Some variant callers such as
MuTect2 and Strelka2 show better performance in sequencing with higher average
sequencing depth and lower coverage [17].
10 Identification of Genetic Variants and de novo Mutations Based on NGS
131