Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species
Major histocompatibility complex (MHC) genes are widely recognised as valuable markers for wildlife genetic studies given their extreme polymorphism and functional importance in fitness-related traits. Newly developed genotyping methods, which rely on the use of next-generation sequencing (NGS), are...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-09-01
|
| Series: | Animals |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2615/12/18/2452 |
| _version_ | 1797491894286024704 |
|---|---|
| author | Ida Svetličić Dean Konjević Elena Bužan Miljenko Bujanić Luka Duniš Sunčica Stipoljev Jelena Martinčić Mihaela Šurina Ana Galov |
| author_facet | Ida Svetličić Dean Konjević Elena Bužan Miljenko Bujanić Luka Duniš Sunčica Stipoljev Jelena Martinčić Mihaela Šurina Ana Galov |
| author_sort | Ida Svetličić |
| collection | DOAJ |
| description | Major histocompatibility complex (MHC) genes are widely recognised as valuable markers for wildlife genetic studies given their extreme polymorphism and functional importance in fitness-related traits. Newly developed genotyping methods, which rely on the use of next-generation sequencing (NGS), are gradually replacing traditional cloning and Sanger sequencing methods in MHC genotyping studies. Allele calling in NGS methods remains challenging due to extreme polymorphism and locus multiplication in the MHC coupled with allele amplification bias and the generation of artificial sequences. In this study, we compared the performance of molecular cloning with Illumina and Ion Torrent NGS sequencing in MHC-DRB genotyping of single-locus species (roe deer) and species with multiple DRB loci (red deer) in an attempt to adopt a reliable and straightforward method that does not require complex bioinformatic analyses. Our results show that all methods work similarly well in roe deer, but we demonstrate non-consistency in results across methods in red deer. With Illumina sequencing, we detected a maximum number of alleles in 10 red deer individuals (42), while other methods were somewhat less accurate as they scored 69–81% of alleles detected with Illumina sequencing. |
| first_indexed | 2024-03-10T00:55:48Z |
| format | Article |
| id | doaj.art-751c60fbb69b41caa983c7e5377d4ee2 |
| institution | Directory Open Access Journal |
| issn | 2076-2615 |
| language | English |
| last_indexed | 2024-03-10T00:55:48Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Animals |
| spelling | doaj.art-751c60fbb69b41caa983c7e5377d4ee22023-11-23T14:43:08ZengMDPI AGAnimals2076-26152022-09-011218245210.3390/ani12182452Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus SpeciesIda Svetličić0Dean Konjević1Elena Bužan2Miljenko Bujanić3Luka Duniš4Sunčica Stipoljev5Jelena Martinčić6Mihaela Šurina7Ana Galov8Department of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, CroatiaFaculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, CroatiaEnvironmental Protection College, Trg mladosti 7, 3320 Velenje, SloveniaFaculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, CroatiaFaculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Glagoljaška 8, 6000 Koper, SloveniaFaculty of Agriculture, University of Zagreb, Svetošimunska cesta 25, 10000 Zagreb, CroatiaDepartment of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, CroatiaDepartment of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, CroatiaDepartment of Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, CroatiaMajor histocompatibility complex (MHC) genes are widely recognised as valuable markers for wildlife genetic studies given their extreme polymorphism and functional importance in fitness-related traits. Newly developed genotyping methods, which rely on the use of next-generation sequencing (NGS), are gradually replacing traditional cloning and Sanger sequencing methods in MHC genotyping studies. Allele calling in NGS methods remains challenging due to extreme polymorphism and locus multiplication in the MHC coupled with allele amplification bias and the generation of artificial sequences. In this study, we compared the performance of molecular cloning with Illumina and Ion Torrent NGS sequencing in MHC-DRB genotyping of single-locus species (roe deer) and species with multiple DRB loci (red deer) in an attempt to adopt a reliable and straightforward method that does not require complex bioinformatic analyses. Our results show that all methods work similarly well in roe deer, but we demonstrate non-consistency in results across methods in red deer. With Illumina sequencing, we detected a maximum number of alleles in 10 red deer individuals (42), while other methods were somewhat less accurate as they scored 69–81% of alleles detected with Illumina sequencing.https://www.mdpi.com/2076-2615/12/18/2452<i>Capreolus capreolus</i><i>Cervus elaphus</i>IlluminaIon Torrentmajor histocompatibility complexnext-generation sequencing |
| spellingShingle | Ida Svetličić Dean Konjević Elena Bužan Miljenko Bujanić Luka Duniš Sunčica Stipoljev Jelena Martinčić Mihaela Šurina Ana Galov Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species Animals <i>Capreolus capreolus</i> <i>Cervus elaphus</i> Illumina Ion Torrent major histocompatibility complex next-generation sequencing |
| title | Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species |
| title_full | Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species |
| title_fullStr | Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species |
| title_full_unstemmed | Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species |
| title_short | Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species |
| title_sort | performance comparison of different approaches in genotyping mhc drb the contrast between single locus and multi locus species |
| topic | <i>Capreolus capreolus</i> <i>Cervus elaphus</i> Illumina Ion Torrent major histocompatibility complex next-generation sequencing |
| url | https://www.mdpi.com/2076-2615/12/18/2452 |
| work_keys_str_mv | AT idasvetlicic performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies AT deankonjevic performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies AT elenabuzan performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies AT miljenkobujanic performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies AT lukadunis performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies AT suncicastipoljev performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies AT jelenamartincic performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies AT mihaelasurina performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies AT anagalov performancecomparisonofdifferentapproachesingenotypingmhcdrbthecontrastbetweensinglelocusandmultilocusspecies |