Additional file 2: of Evaluation of whole-genome sequencing of four Chinese crested dogs for variant detection using the ion proton system

PI chip productivity and per base quality scores. Table describing the yield and base quality of eight sequencing chips. (XLSX 40ย�kb

Genotype data at SNP positions of the <i>IG</i> locus used for identical-by-descent mapping.

Genotype data at SNP positions of the IG locus used for identical-by-descent mapping.</p

Sequence variants (non-reference alleles) in the coding region of <i>LRMDA</i>, <i>ZNF503</i>, and <i>COMTD1</i> detected by sequencing one homozygous <i>IG/IG</i> bird and the corresponding allele frequencies among non-IG <i>(N/-</i>) birds.

Sequence variants (non-reference alleles) in the coding region of LRMDA, ZNF503, and COMTD1 detected by sequencing one homozygous IG/IG bird and the corresponding allele frequencies among non-IG (N/-) birds.</p

Image_4_Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia.tif

IntroductionThe suitability of whole-genome sequencing (WGS) as the sole method to detect clinically relevant genomic aberrations in B-cell acute lymphoblastic leukemia (ALL) was investigated with the aim of replacing current diagnostic methods.MethodsFor this purpose, we assessed the analytical performance of 150 bp paired-end WGS (90x leukemia/30x germline). A set of 88 retrospective B-cell ALL samples were selected to represent established ALL subgroups as well as ALL lacking stratifying markers by standard-of-care (SoC), so-called B-other ALL.ResultsBoth the analysis of paired leukemia/germline (L/N)(n=64) as well as leukemia-only (L-only)(n=88) detected all types of aberrations mandatory in the current ALLTogether trial protocol, i.e., aneuploidies, structural variants, and focal copy-number aberrations. Moreover, comparison to SoC revealed 100% concordance and that all patients had been assigned to the correct genetic subgroup using both approaches. Notably, WGS could allocate 35 out of 39 B-other ALL samples to one of the emerging genetic subgroups considered in the most recent classifications of ALL. We further investigated the impact of high (90x; n=58) vs low (30x; n=30) coverage on the diagnostic yield and observed an equally perfect concordance with SoC; low coverage detected all relevant lesions.DiscussionThe filtration of the WGS findings with a short list of genes recurrently rearranged in ALL was instrumental to extract the clinically relevant information efficiently. Nonetheless, the detection of DUX4 rearrangements required an additional customized analysis, due to multiple copies of this gene embedded in the highly repetitive D4Z4 region. We conclude that the diagnostic performance of WGS as the standalone method was remarkable and allowed detection of all clinically relevant genomic events in the diagnostic setting of B-cell ALL.</p

Genotype distribution of the 2-bp insertion in <i>COMTD1</i> associated with the Inhibitor of Gold (IG) phenotype in different populations sorted by phenotype.

Genotype distribution of the 2-bp insertion in COMTD1 associated with the Inhibitor of Gold (IG) phenotype in different populations sorted by phenotype.</p

Illustration of plumage phenotypes associated with different genotypes at the <i>Inhibitor of gold</i> locus in chicken on different genetic backgrounds.

The birds in (A) and (B) carry the bottom recessive wheaten allele (e) at the MC1R locus and shows red pheomelanin-based pigmentation. The birds in (C) and (D) carry the brown allele (eb) at the same locus that allows expression of both eumelanin and pheomelanin and IG dilution is apparent as regards pheomelanin pigmentation. (A) and (B) depict F2 birds from the mapping pedigree with the wild-type phenotype or the recessive IG phenotype (IG/IG), respectively. (C) and (D) depict two IG/IG birds from the Lemon Millefleur Sabelpoot (Fig 1C) and Sebright-Lemon (Fig 1D) breeds, respectively. Photo by Michèle Tixier-Boichard (A and B) and C and D were taken by Nicolas Bruneau, INRAE (C and D).</p

CRISPR/Cas9 mediated inactivation of <i>Comtd1</i> in B16F10 cells.

(A) Schematic description of CRISPR/Cas9 mediated inactivation of murine Comtd1. Black bars indicate the target sites of two gRNAs in the exons of Comtd1. Yellow arrow indicates a 236bp deletion introduced by paired sgRNAs. The primer pair indicated by red arrows is used for amplifying genomic DNA. (B) Six colonies retrieved from Comtd1 knockout in B16F10 cells. The PAM sequence is in red. Dash (-) indicate deleted nucleotides. The top one is wild type and the others are KO clones, of which, three lines (KO1, KO2, KO3) were generated using sgRNA1, one line was generated using sgRNA2, and the last two lines carrying 236bp deletion were generated by the sgRNA pair. (C) Western blot analysis of whole-cell lysates prepared from six COMTD1 knockout clones and two WT cell lines using antibodies against COMTD1 and the control β-actin. (D) Quantitative RT-PCR analysis of Comtd1 expression in KO and WT cell lines. Date are presented as mean ± SD (n = 3 biological replicates). (E) Cell growth curve of KO (grey) and WT (black) were recorded by the Incucyte Zoom live-cell imaging system and data are expressed as cell confluence (%; mean ± SEM, n = 6 in KO, n = 3 in WT).</p

Table_4_Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia.xlsx

IntroductionThe suitability of whole-genome sequencing (WGS) as the sole method to detect clinically relevant genomic aberrations in B-cell acute lymphoblastic leukemia (ALL) was investigated with the aim of replacing current diagnostic methods.MethodsFor this purpose, we assessed the analytical performance of 150 bp paired-end WGS (90x leukemia/30x germline). A set of 88 retrospective B-cell ALL samples were selected to represent established ALL subgroups as well as ALL lacking stratifying markers by standard-of-care (SoC), so-called B-other ALL.ResultsBoth the analysis of paired leukemia/germline (L/N)(n=64) as well as leukemia-only (L-only)(n=88) detected all types of aberrations mandatory in the current ALLTogether trial protocol, i.e., aneuploidies, structural variants, and focal copy-number aberrations. Moreover, comparison to SoC revealed 100% concordance and that all patients had been assigned to the correct genetic subgroup using both approaches. Notably, WGS could allocate 35 out of 39 B-other ALL samples to one of the emerging genetic subgroups considered in the most recent classifications of ALL. We further investigated the impact of high (90x; n=58) vs low (30x; n=30) coverage on the diagnostic yield and observed an equally perfect concordance with SoC; low coverage detected all relevant lesions.DiscussionThe filtration of the WGS findings with a short list of genes recurrently rearranged in ALL was instrumental to extract the clinically relevant information efficiently. Nonetheless, the detection of DUX4 rearrangements required an additional customized analysis, due to multiple copies of this gene embedded in the highly repetitive D4Z4 region. We conclude that the diagnostic performance of WGS as the standalone method was remarkable and allowed detection of all clinically relevant genomic events in the diagnostic setting of B-cell ALL.</p

Primer sequences used in this paper.

The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin.</div

Table_1_Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia.xlsx

IntroductionThe suitability of whole-genome sequencing (WGS) as the sole method to detect clinically relevant genomic aberrations in B-cell acute lymphoblastic leukemia (ALL) was investigated with the aim of replacing current diagnostic methods.MethodsFor this purpose, we assessed the analytical performance of 150 bp paired-end WGS (90x leukemia/30x germline). A set of 88 retrospective B-cell ALL samples were selected to represent established ALL subgroups as well as ALL lacking stratifying markers by standard-of-care (SoC), so-called B-other ALL.ResultsBoth the analysis of paired leukemia/germline (L/N)(n=64) as well as leukemia-only (L-only)(n=88) detected all types of aberrations mandatory in the current ALLTogether trial protocol, i.e., aneuploidies, structural variants, and focal copy-number aberrations. Moreover, comparison to SoC revealed 100% concordance and that all patients had been assigned to the correct genetic subgroup using both approaches. Notably, WGS could allocate 35 out of 39 B-other ALL samples to one of the emerging genetic subgroups considered in the most recent classifications of ALL. We further investigated the impact of high (90x; n=58) vs low (30x; n=30) coverage on the diagnostic yield and observed an equally perfect concordance with SoC; low coverage detected all relevant lesions.DiscussionThe filtration of the WGS findings with a short list of genes recurrently rearranged in ALL was instrumental to extract the clinically relevant information efficiently. Nonetheless, the detection of DUX4 rearrangements required an additional customized analysis, due to multiple copies of this gene embedded in the highly repetitive D4Z4 region. We conclude that the diagnostic performance of WGS as the standalone method was remarkable and allowed detection of all clinically relevant genomic events in the diagnostic setting of B-cell ALL.</p