minimum depth, whether the variant is in a low-complexity region (i.e., a highly
repetitive region that may increase alignment errors), and more.
Box: Genome Assembly
If there is no reference genome available for our species of interest, it may be worth
trying to create one from scratch. To do this, DNA fragments of the targeted species
are sequenced in high quantity, resulting in sequenced reads that theoretically cover
the entire genome. Reads are aligned and merged based on their overlapping
nucleotides, assembling long DNA sequences. When the order of bases is known
to a high-confidence level, this genomic sequence will be named a “contig.” Multiple
contigs can be assembled together to form a scaffold based on paired read information. A scaffold is a portion of the genome sequences composed of contigs but which
might contain gaps in between them.
There are various tools to close gaps between scaffolds. Scaffolds can then be
joined together to form a chromosome. Despite how easy this may sound, genome
assembly has its difficulties and it can vary between one organism to another (for
example, an uneven representation of the genome due to sequencing sensitivity to
GC bias, which can cause gaps between scaffolds) [39].
Further Reading
The GATK blog: https://software.broadinstitute.org/gatk/documentation/article.php?id=
4148
• Goldstein DB, Allen A, Keebler J, Margulies EH, Petrou S, Petrovski S, et al. Sequencing studies in human genetics: design and interpretation. Nat Rev Genet. 2013 Jul;14
(7):460–70.
• Deciphering Developmental Disorders Study. Large-scale discovery of novel genetic
causes of developmental disorders. Nature. 2015 Mar 12;519(7542):223–8.
• MacArthur DG, Manolio TA, Dimmock DP, Rehm HL, Shendure J, Abecasis GR, et al.
Guidelines for investigating causality of sequence variants in human disease. Nature.
2014 Apr 24;508(7497):469–76.
Somatic variant callers
Mutect2. (Bayesian) Cibulskis, Kristian, Michael S. Lawrence, Scott L. Carter, Andrey
Sivachenko, David Jaffe, Carrie Sougnez, Stacey Gabriel, Matthew Meyerson, Eric S.
Lander, and Gad Getz. “Sensitive Detection of Somatic Point Mutations in Impure and
Heterogeneous Cancer Samples.” Nature Biotechnology. 2013;31(3):213–19.
GATK HaplotypeCaller (Bayesian). McKenna, Aaron, Matthew Hanna, Eric Banks,
Andrey Sivachenko, Kristian Cibulskis, Andrew Kernytsky, Kiran Garimella, et al. “The
138
P. Basurto-Lozada et al.
repetitive region that may increase alignment errors), and more.
Box: Genome Assembly
If there is no reference genome available for our species of interest, it may be worth
trying to create one from scratch. To do this, DNA fragments of the targeted species
are sequenced in high quantity, resulting in sequenced reads that theoretically cover
the entire genome. Reads are aligned and merged based on their overlapping
nucleotides, assembling long DNA sequences. When the order of bases is known
to a high-confidence level, this genomic sequence will be named a “contig.” Multiple
contigs can be assembled together to form a scaffold based on paired read information. A scaffold is a portion of the genome sequences composed of contigs but which
might contain gaps in between them.
There are various tools to close gaps between scaffolds. Scaffolds can then be
joined together to form a chromosome. Despite how easy this may sound, genome
assembly has its difficulties and it can vary between one organism to another (for
example, an uneven representation of the genome due to sequencing sensitivity to
GC bias, which can cause gaps between scaffolds) [39].
Further Reading
The GATK blog: https://software.broadinstitute.org/gatk/documentation/article.php?id=
4148
• Goldstein DB, Allen A, Keebler J, Margulies EH, Petrou S, Petrovski S, et al. Sequencing studies in human genetics: design and interpretation. Nat Rev Genet. 2013 Jul;14
(7):460–70.
• Deciphering Developmental Disorders Study. Large-scale discovery of novel genetic
causes of developmental disorders. Nature. 2015 Mar 12;519(7542):223–8.
• MacArthur DG, Manolio TA, Dimmock DP, Rehm HL, Shendure J, Abecasis GR, et al.
Guidelines for investigating causality of sequence variants in human disease. Nature.
2014 Apr 24;508(7497):469–76.
Somatic variant callers
Mutect2. (Bayesian) Cibulskis, Kristian, Michael S. Lawrence, Scott L. Carter, Andrey
Sivachenko, David Jaffe, Carrie Sougnez, Stacey Gabriel, Matthew Meyerson, Eric S.
Lander, and Gad Getz. “Sensitive Detection of Somatic Point Mutations in Impure and
Heterogeneous Cancer Samples.” Nature Biotechnology. 2013;31(3):213–19.
GATK HaplotypeCaller (Bayesian). McKenna, Aaron, Matthew Hanna, Eric Banks,
Andrey Sivachenko, Kristian Cibulskis, Andrew Kernytsky, Kiran Garimella, et al. “The
138
P. Basurto-Lozada et al.