Marco Savarese
Long-read DNA and RNA sequencing improves analyzing large and complex genes: the example of TTN and NEB
Autori
- RAFAELA OWUSU (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – MSC)
- ELLA NIPPALA (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – MSC)
- ALI OGHABIAN (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – PHD)
- MARIA FRANCESCA DI FEO (DEPARTMENT OF NEUROSCIENCE, REHABILITATION, OPHTHALMOLOGY, GENETICS, AND MATERNAL AND CHILD HEALTH (DINOGMI), UNIVERSITY OF GENOA, GENOVA, ITALY – MD)
- MEHARIJI ARUMILLI (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – PHD)
- DAVID GOMEZ ANDRES (HOSPITAL UNIVERSITARI VALL D’HEBRON, BARCELONA SPAIN – MD)
- LYDIA SAGAT (RADBOUD UNIVERSITY MEDICAL CENTER, DEPARTMENT OF HUMAN GENETICS, RESEARCH INSTITUTE FOR MEDICAL INNOVATION, NIJMEGEN, NETHERLANDS – PHD)
- VILMA LOTTA LEHTOKARI (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – PHD)
- JESSICA CAMACHO SORIANO (HOSPITAL UNIVERSITARI VALL D’HEBRON, BARCELONA SPAIN – MD)
- MARIA ANGELES SANCHEZ DURAN (HOSPITAL UNIVERSITARI VALL D’HEBRON, BARCELONA SPAIN – MD)
- FRANCINA MUNELL (HOSPITAL UNIVERSITARI VALL D’HEBRON, BARCELONA SPAIN – MD)
- KATARINA PELIN (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – PHD)
- PETER HACKMAN (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – PHD)
- BJARNE UDD (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – MD)
- MARCO SAVARESE (FOLKHALSAN RESEARCH CENTER, HELSINKI, FINLAND – PHD)
Presentatore
MARCO SAVARESE
Modalità
Poster Session
Abstract
“Short-read next-generation sequencing (SRS) has been an effective method for genome and transcriptome analysis. Gene panels, exome and genome sequencing have contributed to detecting disease-causing variants. Similarly, short-read RNA sequencing has been thoroughly used to identify and characterize causative variants and study expression and splicing patterns of disease genes. However, SRS technical limitations hamper a proper analysis of complex regions, such as highly repetitive regions in titin (TTN) and nebulin (NEB), and do not allow characterizing long isoforms.
Long-read sequencing (LRS) methods have been shown to overcome most of these technical issues. We hypothesized that DNA LRS could improve variant detection in TTN and NEB repetitive regions and RNA LRS could provide further insights into exon usage and isoform expression in pre- and post-natal muscle samples.
We designed a targeted LRS panel for analyzing the entire genomic sequence of 26 distal myopathy genes, including TTN and NEB. We analyzed sixty DNA samples using PacBio SMRT sequencing and proved that LRS is more effective than SRS when studying TTN and NEB triplicate regions, although still presenting main challenges.
We also performed long-read-isoform sequencing (Iso-Seq) on five different skeletal muscles and one fetal heart from a 21-week fetus, which was aborted for reasons other than a skeletal muscle disease. Iso-Seq was effective in providing sequencing data for TTN, allowing us to map splicing events along different isoforms, but failed to help characterizing NEB isoforms.
LRS is a promising method for DNA and RNA sequencing, although it still presents technical and bioinformatic challenges.”