DataSheet_1_Proteomics data in vitiligo: a scoping review.pdf

An unbiased screening of which proteins are deregulated in vitiligo using proteomics can offer an enormous value. It could not only reveal robust biomarkers for detecting disease activity but can also identify which patients are most likely to respond to treatments. We performed a scoping review searching for all articles using proteomics in vitiligo. Eight manuscripts could be identified. Unfortunately, very limited overlap was found in the differentially expressed proteins between studies (15 out of 272; 5,51%) with variable degrees of the type of proteins and a substantial variety in the prevalence of acute phase proteins (range: 6-65%). Proteomics research has therefore brought little corroborating evidence on which proteins are differentially regulated between vitiligo patients and healthy controls or between active and stable vitiligo patients. While a limited patient size is an obvious weakness for several studies, an incomplete description of patient characteristics is an unfortunate and avoidable shortcoming. Additionally, the variations in the used methodology and analyses may further contribute to the overall observed variability. Nonetheless, more recent studies investigating the response to treatment seem to be more robust, as more differentially expressed proteins that have previously been confirmed to be involved in vitiligo were found. The further inclusion of proteomics analyses in clinical trials is recommended to increase insights into the pathogenic mechanisms in vitiligo and identify reliable biomarkers or promising drug targets. A harmonization in the study design, reporting and proteomics methodology could vastly improve the value of vitiligo proteomics research.</p

Enrichment and sequencing workflow adapted from the ‘Cas9 targeted sequencing’ protocol from ONT.

Two different pools of gRNAs were made. Pool A only contains gRNAs that cut upstream and downstream the CYP2D6-CYP2D7 locus (gRNA7, gRNA1, gRNA8, gRNA2, gRNA5, gRNA6), Pool B also contains a gRNA that cuts between CYP2D6 and CYP2D7 (gRNA4). After dephosphorylation of the genomic DNA, half of the DNA was cleaved by the RNP with the gRNAs of Pool A, and the other half was cleaved by the RNP with the gRNAs of Pool B. After cleavage, the adaptors were ligated at the cleavage site. Next, the two pools were mixed again and purified with AMPure XP beads. The NA12878 libraries were sequenced on a Flongle (NAF) and on a MinION flow cell. The HG01190 and GM19785 libraries were only sequenced on a MinION flow cell. On the runs using a MinION flow cell, half of the pores were controlled by the adaptive sequencing software (NAnCATS-AS, HGnCATS-AS, and GMnCATS-AS), and the other half sequenced conventionally (NAnCATS, HGnCATS, and GMnCATS). *: The NA12878 libraries were used for preliminary optimization purposes and were created with only one pool containing 8 (NAF) or 9 gRNAs (NAnCATS-AS and NAnCATS).</p

Workflow of the in-house developed CoLoRGen pipeline, which combines large structural and small variant calling.

A: The basecalled reads are mapped against the human reference genome GRCh38 (black). Reads are split into the two alleles based on the small variants (yellow and orange stars). Clipping ends of the reads (red) are cut in-silico and mapped again to the reference genome (green). B: The reference is adapted based on the breakpoints of the clipping ends in the DNA under study (grey). Reads of alleles 1 and 2 are mapped against their respective adjusted reference sequence to create a first consensus sequence. C: The reference sequences are further adjusted by mapping all the previously mapped reads to end up with a final consensus sequence. D: The GRCh38 sequences of the CYP2D6 and CYP2D7 genes are mapped against the final consensus sequences. The GRCh38 gene or fragment containing the least mismatches (red stars) is assigned to the corresponding gene or fragment of the consensus sequence, resulting in the determination of the corresponding genes and hybrids. Finally, star-alleles can be assigned based on the determined variants.</p

S1 Text -

Table A: in Overview of the used guide RNAs (gRNAs). Table B: Comparison of small SNV and INDEL variant detection of the Medaka Variant pipeline and the new CoLoRGen tool in the NA12878 DNA sample. Reference: Krusche et al. [36]. Table C: Comparison of structural variant detection of different state-of-the-art structural variant tools and the new CoLoRGen tool in the NA12878, HG01190 and GM19785 DNA samples. For each tool the number of deletions and insertions are given. Between parentheses the length of each variant is given. Green: correctly detected structural variant; red: incorrectly detected structural variant; orange: multiple overlapping structural variants are detected although only one variant is present in the reference. Reference: Get-RM studies [15,16]. †: the found regions show overlap. (DOCX)</p

Star-alleles in literature references and star-alleles assigned by the CoLoRGen pipeline.

Reference star-alleles were obtained from Krusche et al. [36] and the Get-RM studies [15,16]. The depths mentioned below the genes are the generated average depths on that position of the locus. ¥The *68 allele was only detected when TaqMan-based genotyping was combined with CNV and structural variant detection using quantitative multiplex PCR and LR-PCR validation. Therefore, the Get-RM consensus star-allele only mentions the *68 allele in brackets. Note: even when depths below the minimal 16X depth for reliable small variant calling were obtained, correct star-alleles could be assigned.</p

CoLoRGen detected four additional small variants in the GM19785 cell line that are not present in the sub-allele definitions.

The three deletions were located in homopolymeric regions and the SNV is a silent mutation. (PDF)</p

Femto pulse profiles of the used DNA samples.

The x-axis represents the DNA fragment size (non-linear scale). The y-axis represents the fluorescent signal proportional to the amount of DNA. A: NA12878, B:HG01190, and C:GM19785. (PDF)</p

General sequencing results of the nCATS-enriched NA12878, HG01990, and GM19785 libraries.

General sequencing results of the nCATS-enriched NA12878, HG01990, and GM19785 libraries.</p

Reads of the HG01190 DNA, sequenced on a MinION flow cell, mapped on the GRCh38 reference genome.

The HGcombined dataset was used to generate this figure, which is the dataset containing both the positively selected reads from the AS pores and all the reads from the conventionally sequencing pores. The positions of the gRNAs are indicated with vertical lines and the sequencing direction is indicated with arrows on top of the vertical lines. Reads are split by allele, and gray reads are clipping ends that were cut in-silico and mapped separately. The cut position of the clipping ends indicating the start and end position of the deletion and insertion are indicated in gray. (PDF)</p

Representation of the called and phased small variants (SNVs and INDELs) in the CYP2D6 and CYP2D7 genes of the NA_combined library.

The truth set composed by Krusche et al. [36] was used as reference (Ref). Green lines represent concordant calls (true-positives compared to the truth set), which are correctly called and phased variants compared to the reference; orange lines represent discordant calls (false-positives compared to the truth set). Note: multiple variants next to each other are visually represented by thicker lines.</p