Naslov
Experiment adjusted bioinformatics workflow reduces genotyping errors in polymorphic multilocus MHC systems

Autori
Arbanasić, Haidi ; Križanović, Krešimir ; Svetličić, Ida ; Bugarski, Dejan ; Mirčeta, Jovan ; Konjević, Dean

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Abstract book / - , 2022, 33-33

Skup
5th European Conservation Genetics Meeting

Mjesto i datum
Edinburgh, Ujedinjeno Kraljevstvo, 30.08.2022. - 01.09.2022

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
red deer, NGS, DRB, alleles

Sažetak
The Major Histocompatibility Complex (MHC) is highly polymorphic genetic region involved in immune response and significant indicator of adaptively important genetic diversity. Due to extensive variability represented by large number of alleles in population and locus duplications, accurate genotyping of MHC genes may be challenging. Next Generation Sequencing (NGS) technologies are particularly applicable for the genotyping of highly polymorphic, multilocus MHC systems. Here, we analyzed 227-bp part of exon 2 of the MHC class II DRB gene in 70 red deer (Cervus elaphus) individuals from Serbia, using Illumina platform. The major challenge in analyzing NGS data is to separate artefacts that include polymerase nucleotide misincorporation and chimeras, from true alleles. Namely, artefacts largely outnumber true alleles, which may be under-amplified due to primer mismatches. To identify alleles, we developed a workflow adjusted to our experiment, where 60% of individuals had amplicon replicates. We identified 26 DRB alleles, whose population frequency ranged from 0.4% to 11.9%, and found 1-6 alleles per individual. Within-amplicon allele frequency ranged from 1.2% to 79%, while the most frequent artefact was represented by 7% of amplicon reads. Two alleles were characterized by low intra-amplicon frequencies, suggesting low amplification efficiency. Further, we conducted genotyping analysis by Amplisas tool (Amplicon Sequencing Assignment) using default Illumina parameters. In comparison to our workflow, Amplisas dropped 19 allele assignments, all connected to low amplicon frequency or small number of reads. We conclude that lab developed workflow reduces genotype calling errors, particularly for extremely polymorphic genes, such as MHC class II DRB.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA