Izvorni znanstveni članak
https://doi.org/10.3325/cmj.2024.65.180
Non-invasive prenatal testing of beta-hemoglobinopathies using next generation sequencing, in-silico sequence size selection, and haplotyping
Henry A. Erlich
; UCSF Benioff Children’s Hospital Oakland Research Institute, Oakland, CA, USA
*
Lily Ko
; Department of Pediatrics, University of California, San Francisco, Oakland, CA, USA
Jiyae Lee
; Department of Pediatrics, University of California, San Francisco, Oakland, CA, USA
Katrina Eaton
; Department of Pediatrics, University of California, San Francisco, Oakland, CA, USA
Cassandra D. Calloway
; UCSF Benioff Children’s Hospital Oakland Research Institute, Oakland, CA, USA
Ashutosh Lal
; Department of Pediatrics, University of California, San Francisco, Oakland, CA, USA
Reena Das
; Post-Graduate Institute of Medical Education and Research, Chandigarh, India
Manu Jamwal
; Post-Graduate Institute of Medical Education and Research, Chandigarh, India
Christian Lopez-Pena
; Department of Pediatrics, University of California, San Francisco, Oakland, CA, USA
Steven J. Mack
; Department of Pediatrics, University of California, San Francisco, Oakland, CA, USA
* Dopisni autor.
Sažetak
Aim To develop a non-invasive prenatal test for beta-hemoglobinopathies based on analyzing maternal plasma by
using next generation sequencing.
Methods We applied next generation sequencing (NGS)
of maternal plasma to the non-invasive prenatal testing
(NIPT) of autosomal recessive diseases, sickle cell disease
and beta-thalassemia. Using the Illumina MiSeq, we sequenced plasma libraries obtained via a Twist Bioscience
probe capture panel covering 4 Kb of chromosome 11,
including the beta-globin (HBB) gene and >450 genomic
single-nucleotide polymorphisms (SNPs) used to estimate
the fetal fraction (FF). The FF is estimated by counting paternally transmitted allelic sequence reads present in the
plasma but absent in the mother. We inferred fetal betaglobin genotypes by comparing the observed mutation
(Mut) and reference (Ref ) read ratios to those expected for
the three possible fetal genotypes (Mut/Mut; Mut/Ref; Ref/
Ref ), based on the FF.
Results We bioinformatically enriched the FF by excluding reads over a specified length via in-silico size selection
(ISS), favoring the shorter fetal reads, which increased fetal genotype prediction accuracy. Finally, we determined
the parental HBB haplotypes, which allowed us to use the
read ratios observed at linked SNPs to help predict the fetal genotype at the mutation site(s). We determined HBB
haplotypes via Oxford Nanopore MinION sequencing of a
2.2 kb amplicon and aligned these sequences using Soft
Genetics’ NextGENe LR software.
Conclusion The combined use of ISS and HBB haplotypes
enabled us to correctly predict fetal genotypes in cases
where the prediction based on variant read ratios alone
was incorrect
Ključne riječi
Hrčak ID:
331953
URI
Datum izdavanja:
16.6.2024.
Posjeta: 0 *