A total of 1400 subjects of childbearing age who came to our hospital for carrier screening during July 2023 to December 2023 (male to female ratio was about 1:1) and seven samples from one family were enrolled in this study. Informed consent was obtained from all participants or their legal guardians involved in the study.

DNA was extracted and measured using the Blood Genomic DNA Mini Kit (Cwbio, Beijing, China) and NanoDrop spectrophotometer, respectively. DNA was stored in an environment of -20℃ before use.

The SALSA® MLPA® Probemix P060-B2 SMA Carrier (MRC-Holland, Amsterdam, The Netherlands) was used in this study. The kit was completed according to the manufacturer’s instructions. After denaturation, hybridization, probe connection, and PCR, a 3500xL Dx genetic analyzer (Applied Biosystems, Foster City, CA) was used for detection. The test data were imported into Coffalyser (v.220513, MRC Holland, Amsterdam, The Netherlands) for further analysis.

The Motor Neuron Survival Gene 1 (SMN1) Exon Deletion Detection Kit (Wuse Shi Medical Technology, Shanghai, China) was used for the qPCR assay in this study. Exons 7 and 8 of SMN1 were amplified and relatively quantified. And chemical blocking was used to control the effect of SMN2 on the detection results. The test was performed on the Hongshi SLAN96S real-time fluorescence analysis system (Hongshi, Shanghai, China) following the procedure: 95℃ for 10 min, 40 cycles of 95℃ for 15 s and 58℃ for 60 s. Fluorescence was collected after each cycle. In this study, two samples with two-copy SMN1 were added to each assay as a reference, and the relative copy number could be roughly analyzed using the 2−ΔΔCq method.

The full-length sequence of SMN1/2 was determined by the method named CASMA based on the LRS (the third-generation sequencing, TGS) as previously described [17]. Briefly, the full-length sequence of the SMN1 and SMN2 genes was amplified (KOD FX Neo Polymerase, TOYOBO, Osaka, Japan) and ligated by a unique hairpin barcode adapter to form a dumbbell-shaped pre-library. Exonuclease (Enzymatics, Beverly, MA) was then added to remove failed DNA ligations. After purification and quantification, equal mass was pooled to form single-molecule real-time dumbbell (SMRTbell) libraries. SMRTbell libraries were prepared using the Sequel II Binding Kit 3.2 (Pacific Biosciences, CA, US) and then sequenced on the Sequel IIe platform (Pacific Biosciences, CA, US) for 30 h using the cyclic consensus sequencing (CCS) mode.

Raw subreads of each sample were debarcoded and aligned to the hg38 reference using lima (in the Pbbioconda package, smrtlink 10.1.0.119588, Pacific Biosciences) and pbmm2 (version 1.5.0), respectively. SMN1 and SMN2 genes were differentiated by c.840. For haplotype analysis, each CCS read was aligned with the internal reference gene to obtain all SNPs using FreeBayes version 1.3.4 (Biomatters, Inc.,San Diego, CA). Only CCS reads whose SNPs frequencies between 20% and 80% were retained for haplotype determination. These CCS reads were recursively divided into two groups by SNPs until no further division was possible [17]. Each final group was a specific haplotype, and the number of reads per haplotype was counted. The copy numbers of SMN1 and SMN2 were determined using a Poisson distribution-based caller with the haplotype numbers and read count as inputs [16]. CCS reads of representative samples were displayed in the Integrated Genomics Viewer (IGV) to show the different haplotypes of SMN1 and SMN2.

STR analysis was used to determine the relationships among family members. Multiple STRs were analyzed using the Goldeneye™ DNA ID System 20 A (Beijing PeopleSpot Inc, Beijing, China) with 19 target loci of D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, and FGA. Sex was determined using characterized sequences in Amelogenin. DNA from each sample was amplified using the Applied Biosystems Veriti instrument (Applied Biosystems, Foster City, CA), and was sequenced on the 3500xL Dx genetic analyzer (Applied Biosystems, Foster City, CA). Finally, the assay data were imported into the GeneMapper® ID-X (Applied Biosystems, Foster City, CA) to complete the analysis.