Applying targeted long-read sequencing to assess an expanded repeat in C9orf72

Presented at: CRISPR 2020
C.E. Credits: P.A.C.E. CE Florida CE
Speaker

Abstract

A repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) represents the most common genetic cause of two fatal neurodegenerative diseases: frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To determine the length of this repeat expansion, we used No-Amp sequencing (Pacific Biosciences [PacBio]), a targeted amplification-free long-read sequencing technology. In total, we examined 28 fully characterized C9orf72 expansion carriers for whom cerebellar tissue was available. We obtained more than 3500 circular consensus sequencing (CCS) reads, including 814 reads that spanned the C9orf72 repeat expansion. Repeat lengths obtained using No-Amp sequencing significantly correlated with those obtained using Southern blotting. Importantly, we demonstrated that smaller expansions are associated with prolonged survival after onset. Moreover, we revealed that smaller expansions associate with higher levels of transcripts containing the expansion and of dipeptide repeat (DPR) proteins, a hallmark of C9orf72-linked diseases. These findings suggest that, during transcription, it might be easier to cover small expansions than long expansions, which could explain why we observed elevated levels of expansion-containing transcripts in patients with smaller expansions. Since these expansion-containing transcripts can serve as templates for repeat-associated non-ATG (RAN) translation, this could account for the increased levels of DPR proteins we detected. Investigation of the expansion itself uncovered that its GC-content is high (100%). The expansion appears to be relatively pure, while interruptions are only occasionally encountered (<5%). In conclusion, we have shown that No-Amp sequencing is a powerful tool to examine the length and purity of repeat expansions, which enabled us to identify relevant clinico-pathological associations for C9orf72-related diseases.

Learning Objectives:

1. Learn about methods that can be employed to detect and size C9orf72 repeat expansions

2. Understand that targeted long-read sequencing accurately measures the C9orf72 repeat length and presence of interruptions


Show Resources
You May Also Like
SEP 14, 2021 7:00 AM PDT
Add to Calendar Select one of the following: iCal Google Calendar Outlook Calendar Yahoo Calendar
C.E. CREDITS
SEP 14, 2021 7:00 AM PDT
Add to Calendar Select one of the following: iCal Google Calendar Outlook Calendar Yahoo Calendar
Date: September 14, 2021 Time: 7am PDT, 10am EDT, 4pm CEST A conventional thermal cycler has long been a commodity product in the lab and end-point PCR techniques can be completed almost wit...
MAY 11, 2021 10:00 AM PDT
C.E. CREDITS
MAY 11, 2021 10:00 AM PDT
Date: May 11, 2021 Time: 10:00zm PDT Your samples are some of the most valuable assets in the laboratory. After spending countless hours on extraction and preparation, your conclusions could...
MAR 16, 2021 10:00 AM PDT
C.E. CREDITS
MAR 16, 2021 10:00 AM PDT
Date: March 16, 2021 Time: 10:00am (PST) Scientific progress and breakthroughs today are often too expensive for most institutions to acquire. Each year, the National Institutes of Health (N...
JUN 09, 2021 7:00 AM PDT
C.E. CREDITS
JUN 09, 2021 7:00 AM PDT
Date: June 9, 2021 Time: 09 June 2021, 7am PDT, 10am EDT, 4pm CEST cells with dramatic implications on the validity of past cell culture related research. The fact that at least 509 cell lin...
APR 01, 2021 8:00 AM PDT
C.E. CREDITS
APR 01, 2021 8:00 AM PDT
Date: April 01, 2021 Time: 8:00am (PST), 11:00am (EST) Generating therapeutic antibodies is far more challenging than obtaining antibodies that merely recognize their targets. Engineering po...
MAR 18, 2021 8:00 AM PDT
C.E. CREDITS
MAR 18, 2021 8:00 AM PDT
DATE: March 18, 2021 TIME: 8:00am PDT Sequencing of bulk cells, single cells, and nuclei is opening doors in the understanding of complex biological processes....
Loading Comments...
Show Resources