Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs

Abstract

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends, capturing the target region for sequencing on PacBio's Sequel, Sequel II, or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands. Key features • This protocol is adapted from PacBio's previous "no-amp targeted sequencing utilizing the CRISPR-Cas9 system." • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type, blood, or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio.

Keywords: C9orf72; No amplification; Repeat expansion; Single-molecule sequencing; iPSCs.

Figure 1.. 1% Agarose gel evaluation of genomic DNA quality.

As a quick quality check, we run isolated DNA on a 1% agarose gel prior to initiating CRISPR isolation of the target region. We include only DNA that has high molecular weight (i.e., has no smearing). (A, B) Examples of genomic DNA samples extracted from C9orf72 patient iPSC lines and run on a 1% agarose gel to evaluate sample quality. (A) Samples 1, 2, 4, and 5 (A) and 6 (B) show no smearing and would be acceptable for use in the protocol. Samples 3 (A) and 7 (B) show significant smearing and therefore we recommend being excluded from this protocol. This is a quick quality check. Sample quality can be confirmed with higher accuracy by the Agilent FEMTO Pulse System, the Bio-Rad CHEF Mapper XA Pulsed Field Electrophoresis System, or the Sage Science Pippin Pulse Electrophoresis Power Supply.

Figure 2.. Example of PacBio Sequel II sequencing output.

Waterfall plots of single-molecule sequencing reads. Each horizontal line represents one molecule sequenced, anchored at a non-repeat region 5′ to the C9orf72 repeat expansion. The X-axis shows the nucleotide position compared to the anchor. The Y-axis shows the CCS read count. The grey color represents changes in the sequence other than GGGGCC repeat, which are likely to be a sequencing error. (A) NoAmp sequencing of patient iPSC DNA with two repeats on the normal allele (tall blue tail reaching 300 CCS reads) and approximately 250 repeats on the expanded allele. (B) NoAmp sequencing of patient iPSC DNA showing repeat lengths from 2 to 250 representing mosaicism in this line.