A technical application of quantitative next generation sequencing for chimerism evaluation

Abstract

At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44-amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (<1%). The protocol followed the standard Ion AmpliSeq library preparation and Ion Torrent Personal Genome Machine guidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification.

Simultaneous Monitoring of Mutation and Chimerism Using Next-Generation Sequencing in Myelodysplastic Syndrome

“…We did not detect any donor carrying a clonal hematopoiesis-related mutation [20]. The number of informative SNPs was variable (median 25.5, range 9-41) and higher than that of STR (median 10.5, range [5][6][7][8][9][10][11][12][13][14]. Donor chimerism from NGS analysis highly correlated with results from STR analysis, with r 2 = 0.994 (p < 0.0001) by least-squares analysis (Figure 2a), and all data points were within 3 SDs of the mean % difference on Blant-Altman plot (Figure 2b).…”

“…For NGS chimerism analysis, we reviewed all the identified SNPs as the frequency of heterozygosity in the general Korean population (the Korean reference genome database investigated 1722 Korean individuals). The homozygous and heterozygous alleles were determined by base count frequency of 90-100% and 45-60% [8], respectively. We examined 153 SNPs with a frequency of heterozygosity ranging from 0.2 to 0.8 in the Korean database and selected optimal SNPs for NGS chimerism analysis based on the following criteria: (1) >500 mean read depth, (2) ≤0.2% BE, and (3) <10% measurement error of heterozygous alleles (ME, difference of read count between reference and alternative alleles) [19].…”

“…Real-time quantitative PCR of insertion/deletion markers showed higher sensitivity with a 0.1% threshold compared with that of STR analysis [7]. Recently, a custom NGS chimerism panel using single-nucleotide polymorphism (SNP) markers has been applied to chimerism and showed a high sensitivity of 0.5-1%, which was higher than that of STR analysis [8,9]. Hence, the optimal method for monitoring MRD in MDS both pre-and post-HSCT may require more than one technology [10].…”

Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

“…Second, detection of informative loci is accomplished through massively-parallel sequencing, which provides sequence information for each capture event and allows them to be accurately mapped within the genome, further reducing the measurement of stochastic, off-target captures. These features offer advantages over next generation sequencing approaches that identify chimerism based on single nucleotide polymorphisms (36,37), which are subject to sequencing errors that limit their sensitivity(7,37). …”

Could a chimeric condition be responsible for unexpected genetic syndromes? The role of the single nucleotide polymorphism‐array analysis

“…We discuss SNP array as a technique able to identify chimerism also suggesting which one of the complex mechanisms underlying chimera formation could be responsible (Biesecker & Spinner, 2013). Subsequently, a custom nextgeneration sequence (NGS) panel was used for chimerism quantification (Aloisio et al, 2016).…”