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M. Zaliova et al.
barcodes which enabled us to multiplex them and avoid carry-over contamination. To amplify the ERGdel span- ning region, the same gene specific primers were used as for the PCR, targeting the most frequent type of ERGdel utilizing a common 5’ breakpoint site and one of the five different 3’ breakpoint site clusters. In the AmpliSeq, 1-2 ERG germline region(s) (control amplicons) were co- amplified within the same PCR reaction (Figure 1). This approach enabled us to quantify resulting amplicon libraries and achieve comparable sequencing depth (and thus sensitivity) across all patients, including potentially ERGdel-negative cases without any detectable or quantifi- able ERGdel PCR product after PCR. The sensitivity of
AmpliSeq was tested on a dilution series of positive con- trol; using 1x106 coverage we achieved sensitivity of 0.01%. Two different levels of coverage were used in the AmpliSeq experiments. Fifteen PCR-positive patients were sequenced with standard coverage (2x105 reads per sample), and 18 PCR-positive DUX4r-ALL patients and 26 PCR-negative patients (17 from the DUX4r cohort and 9 from the non-DUX4r B-others) were sequenced with higher target coverage (1x106 reads per sample). The num- ber of ERGdel reads in all but one patient was high (103- 106); in the remaining single patient (UPN-004), who was PCR-negative, we found only 13 ERGdel reads. To assess sensitivity of AmpliSeq used at lower coverage, we per-
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Figure 3. ERGalt expression in acute lymphoblastic leukemia (DUX4r-ALL) strat- ified by ERGdel. ERGalt expression (y-axis: normalized read counts from RNAseq data) is shown for DUX4r-ALL patients stratified by ERGdel according to results of single nucleotide polymorphism array (A), polymerase chain reaction (B) and amplicon sequencing (C). P-values from Mann-Whitney U test are shown. NS: dif- ference not statistically significant.
Figure 4. Total copy number aberrations (CNA) in acute lymphoblastic leukemia (DUX4r-ALL) stratified by ERGdel. The total number of CNA are shown for DUX4r-ALL patients stratified by ERGdel according to results of single nucleotide polymorphism array (A), polymerase chain reaction (B) and amplicon sequencing (C). P-values from Mann-Whitney U test are shown.
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haematologica | 2019; 104(7)