Vega targeted datasets

Consolidate legacy methods into a single assay with 1-day PureTarget prep on the Vega system

Modernize your molecular assays with the PureTarget repeat expansion panel on the Vega benchtop system.This highly accurate long-read sequencing panel lets you consolidate multiple methods with a 1-day prep, 48-sample multiplexing, and the preservation of crucial methylation data. As an amplification-free prep, PureTarget allows you to easily resolve challenging repeats in the FMR1, C9orf72, and RFC1 genes which short reads can’t fully capture. This benchtop panel keeps you in control, getting fast results without relying on third-party sequencing services.

Highlights:

Comprehensive genotyping of the most challenging targets
High on-target coverage enables accurate genotyping

Key metrics

Minimum coverage for 2 μg of input DNA from supported samples types (Nanobind-extracted human blood and cell line) for unexpanded alleles.

Comprehensive genotyping of the most challenging targets

Repeat expansions in the FXN and RFC1 genes occur in high frequency across many human populations, with recent estimates that 1 in ~14 individuals carry pre–mutations at RFC1 and 1 in ~100 carry pre-mutations at FXN (Ibañez et al., 2024). Accurate repeat sizing is important to identify carriers of pathogenic alleles longer than 70 repeats for FXN and longer than 400 repeats at RFC1 (Leitão et al., 2024). Long and accurate HiFi reads make it possible to distinguish among the diverse repeat motifs at RFC1, only some of which are pathogenic (Dominik et al., 2023).

RFC1 expansions

TRVZ plot of NA20752 at RFC1(ital) repeat showing consensus allele for the normal (top) and pathogenic expanded allele (bottom) with phased reads aligned to each consensus sequence. 657 “AAGGG” motifs are observed in the expanded allele with consensus length of 3253 bp. — **TRGT plot**: NA20752 at *RFC1* repeat showing consensus allele for the normal (top) and pathogenic expanded allele (bottom) with phased reads aligned to each consensus sequence. Also, 657 “AAGGG” motifs are observed in the expanded allele with consensus length of 3253 bp.

TRVZ plot of HG01175 at RFC1 repeat showing consensus allele for the normal (top) and pathogenic expanded allele (bottom) with phased reads aligned to each consensus sequence. 393 “AAGGG” motifs are observed in the expanded allele with consensus length of 1948 bp. Note the diversity of repeat motifs which are distinguishable by HiFi reads. — **TRGT plot**: HG01175 at *RFC1* repeat showing consensus allele for the normal (top) and pathogenic expanded allele (bottom) with phased reads aligned to each consensus sequence. Also, 393 “AAGGG” motifs are observed in the expanded allele with consensus length of 1948 bp. Note the diversity of repeat motifs which are distinguishable by HiFi reads.

FXN expansions

	Long allele					Short allele
Sample	Coverage	Observed motif count	Expected motif count	Coverage	Observed motif count	Expected motif count
HM16212	92	471	500	146	8	<30
NA16212	68	515	500	99	8	<30
NA16202	49	817	830	86	8	<30
NA16237	49	699	700	134	8	<30

High on-target coverage enables accurate genotyping

The PureTarget repeat expansion panel enables deep coverage for accurate genotyping of repeat expansions associated with human disease. The table below highlights typical coverage for Coriell samples with known repeat expansions. Note that 2 µg of DNA was prepared with the PureTarget repeat expansion panel of 20 targets, sequenced on the Vega system, and analyzed with TRGT (Dolzhenko et al., 2024) in the SMRT Link PureTarget repeat expansion panel analysis workflow. Full dataset available here.

Sample	Target with repeat expansion	Total reads per sample	Mean target coverage	Coverage of expanded allele	Epanded allele size (bp)
20 gene panel
HM23709	AR	54,252	343	182	147
NA13716	ATN1	40,700	234	88	216
NA13536	ATXN1	39,845	267	132	124
NA06153	ATXN3	64,164	258	114	207
ND14442	C9orf72	16,821	51	4	4563
HM03756	DMPK	52,504	395	282	1305
NA09237	FMR1	36,167	257	113	2683
NA16215	FXN	67,939	245	62	2963
NA13509	HTT	58,870	480	225	252
HM23629	PABPN1	50,569	417	218	27
HG01175	RFC1	53,454	378	57	1948

3 days from sample to insight

Extraction

Start with HMW DNA extracted from human blood or cell lines using Nanobind extraction kits.

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Library prep

Targeting and library prep can be completed in 8 hours. All reagents supplied in the kit.

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Sequencing

Sequence up to 48 samples per Vega SMRT Cell with 24 hour movies and optimized run conditions for PureTarget libraries.

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Data analysis

SMRT Analysis is automatically configured to generate library QC, target enrichment statistics, and tandem repeat genotypes using TRGT (Dolzhenko et al., 2024).

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References

Dolzhenko, E., et al. (2024). Characterization and visualization of tandem repeats at genome scale. Nat Biotechnol. 2024 doi: 10.1038/s41587-023-02057-3.

Dominik, N., et al. (2023). Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis. Brain. 146(12):5060-5069. doi: 10.1093/brain/awad240.

Ibañez, K.,  et al.  (2024) Increased frequency of repeat expansion mutations across different populations. Nature Medicine. https://doi.org/10.1038/s41591-024-03190-5

Leitão, E., et al. (2024). Identification and characterization of repeat expansions in neurological disorders: Methodologies, tools, and strategies. Rev Neurol (Paris). 180(5):383-392. doi: 10.1016/j.neurol.2024.03.005.