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The goals of this study were to (a) ascertain human identity capabilities of DNA obtained from latent fingerprints that have been first environmentally insulted and then developed by the deposition of a columnar thin film (CTF), and (b) to determine if the CTF process and material are detrimental to single nucleotide polymorphism (SNP) analysis. Fingerprints were deposited on five different types of substrates and aged for one day, 7 days or 30 days while being environmentally insulted under one of the four conditions: 16.6 °C and 60% relative humidity (RH) (Condition A), 24.5 °C and 60% RH (Condition B), 35 °C and 67% RH (Condition C) and a cold condition (Condition D). Then CTF technique was then on 59% of these fingerprints. DNA samples from 805 fingerprints were extracted, quantified, subjected to manual library preparation using the Precision ID Identity Panel, and underwent high-throughput sequencing. The Ion S5™ platform was employed to sequence 124 SNP amplicons. SNPs were successfully sequenced from 802/805 samples. Total read depth was consistent across environmental conditions, and majority of samples had 100% profile completeness and 100% concordance. Anecdotally, libraries that were amplified with a higher cycle number had more ‘Major Allele Frequency’ flags compared to samples amplified with 23 cycle numbers, possibly due to stochastic effects. Neither the substrates nor the CTF process and materials inhibit downstream DNA analysis. DNA of low quality and quantity from the chosen samples can be sequenced using the Precision ID Identity Panel on the Ion S5™ platform which performed well, however, a different approach may be needed if spurious alleles are suspected.

Purpose Hearing loss (HL) is the most common sensory disorder in children. Prompt molecular diagnosis may guide screening and management; especially in syndromic cases when HL is the single presenting feature. Exome sequencing (ES)is an appealing diagnostic tool for HL as the genetic causes are highly heterogeneous. Methods ES was performed on a prospective cohort of 43 probands with HL. Sequence data were analyzed for primary and secondary findings. Capture and coverage analysis was performed for genes and variants associated with HL. Results The diagnostic rate using ES was 37.2% compared to 15.8% with clinical HL panel. Secondary findings were discovered in three patients. For 247 genes associated with HL, 94.7% of the exons were targeted for capture and 81.7% of these exons were covered at 20× or greater. Further analysis of 454 randomly selected HL-associated variants showed 89% were targeted for capture and 75% were covered at a read depth of at least 20×. Conclusion ES has an improved yield to clinical testing and may capture diagnoses not initially considered due to subtle clinical phenotypes. Technical challenges were identified, including inadequate capture and coverage of HL genes. Additional considerations of ES include secondary findings, cost, and turnaround time.

One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA. The resulting Penn Precision Panel (PPP) targeted 20 genes containing clinically significant variants relevant to many cancers. One hundred twenty-three samples were analyzed, including 83 solid tumor specimens derived from FFPE. Various input quantities of DNA (0.5-10 ng) were amplified with content-specific PCR primer pools, then sequenced on a MiSeq instrument (Illumina, Inc.) via paired-end, 2 × 186 base pair reads to an average read depth of greater than 6500x. Variants were detected using an in-house analysis pipeline. Clinical sensitivity and specificity were assessed using results from our previously validated solid tumor NGS panel; sensitivity of the PPP is 96.75% (387/400 variants) and specificity is 99.9% (8427/8428 base pairs). Variant allele frequencies (VAFs) are highly concordant across both assays (r = 0.98 p 0.0001). The PPP is a robust, clinically validated test optimized for low-yield solid tumor specimens, capturing a high percentage of clinically relevant variants found by larger commercially available NGS panels while using only 0.5-10 ng of input DNA.

Copy number variation (CNV) is a common phenomenon in the human genome, accounting for a large proportion of structural variations. Accurate detection of CNVs has been regarded as a regular approach for studying cancer mechanisms and evolution and seeking targeted drugs for cancer diagnosis. Next-generation sequencing (NGS) data has provided an unprecedented opportunity for this task. Currently, a large number of methods have been developed for CNV detection by using NGS data. Each of them has its own viewpoint on the characteristics of CNVs associated with the features of NGS data. Due to the intrinsic complexity of CNVs, new methods with a comprehensive viewpoint on the characteristics of CNVs combined with NGS data are required for the detection of various forms of CNVs. In this paper, we propose an intra-hybrid approach, IhybCNV, for the detection of CNVs from NGS data. The central idea of the IhybCNV is that it regards CNVs as outlier events from various viewpoints on the read depth (RD) profile to be analyzed. More precisely, the IhybCNV integrates five individual detectors to calculate five types of outlier scores for each genome segment and then combines the five types of outlier scores into an anomaly score via locally selective combination in parallel outlier ensembles (LSCP). With the anomaly scores of the genome segments, the IhybCNV determines abnormal segments (CNVs) or normal segments by designing a binary clustering model (BCM). The performance of the IhybCNV is evaluated by carrying out simulation studies and real data applications and is compared with five peer methods. The experimental results demonstrate that the IhybCNV obtains a higher F1-score value than the other methods. Thus, the IhybCNV could be expected as a supplementary to current methods and might become a promising tool for the detection of genome mutations.

High throughput single-cell RNA sequencing (sc-RNAseq) has become a frequently used tool to assess immune cell function and heterogeneity. Recently, the combined measurement of RNA and protein expression by sequencing was developed, which is commonly known as CITE-Seq. Acquisition of protein expression data along with transcriptome data resolves some of the limitations inherent to only assessing transcript, but also nearly doubles the sequencing read depth required per single cell. Furthermore, there is still a paucity of analysis tools to visualize combined transcript-protein datasets. Here, we describe a novel targeted transcriptomics approach that combines analysis of over 400 genes with simultaneous measurement of over 40 proteins on more than 25,000 cells. This targeted approach requires only about 1/10 of the read depth compared to a whole transcriptome approach while retaining high sensitivity for low abundance transcripts. To analyze these multi-omic transcript-protein datasets, we adapted One-SENSE for intuitive visualization of the relationship of proteins and transcripts on a single-cell level.

Background/objectives The UBR1 gene encodes the enzyme ubiquitin-protein ligase E3 component n-recognin 1. Loss-of-function mutations in the UBR1 gene cause Johanson-Blizzard syndrome, which involves pancreatic exocrine insufficiency. No previous studies have examined an association of UBR1 variants with pancreatitis, in part due to the large size of the gene. This study aimed to clarify whether UBR1 variants are associated with chronic pancreatitis (CP) by the application of targeted next generation sequencing. Methods Exon sequences of the UBR1 gene from 389 Japanese patients with CP (188 idiopathic, 172 alcoholic, 20 hereditary, 9 familial) were captured by the HaloPlex target enrichment technology and subjected to next generation sequencing. Results Ninety nine point two % of the coding regions of the UBR1 gene could be sequenced by ≥ 20 reads with a mean read depth of 595 and a median depth of 399. Fifteen non-synonymous variants including three novel ones [c.4514T > C (p.I1505T), c.4828C > G (p.H1610D) and c.4856A > T (p.D1619V)] and two synonymous variants were identified in the exonic regions. The frequency of any non-synonymous or synonymous variants was not different between the patients with CP and controls. Conclusions Variants in the UBR1 gene were not associated with CP in Japan.

The use of massively parallel sequencing of maternal cfDNA for non-invasive prenatal testing (NIPT) of aneuploidy is widely available. Recently, the scope of testing has increased to include selected subchromosomal abnormalities, but the number of samples reported has been small. We developed a calling pipeline based on a segmentation algorithm for the detection of these rearrangements in maternal plasma. The same read depth used in our standard pipeline for aneuploidy NIPT detected 15/18 (83%) samples with pathogenic rearrangements > 6 Mb but only 2/10 samples with rearrangements < 6 Mb, unless they were maternally inherited. There were two false-positive calls in 534 samples with no known subchromosomal abnormalities (specificity 99.6%). Using higher read depths, we detected 29/31 fetal subchromosomal abnormalities, including the three samples with maternally inherited microduplications. We conclude that test sensitivity is a function of the fetal fraction, read depth, and size of the fetal CNV and that at least one of the two false negatives is due to a low fetal fraction. The lack of an independent method for determining fetal fraction, especially for female fetuses, leads to uncertainty in test sensitivity, which currently has implications for this technique’s future as a clinical diagnostic test. Furthermore, to be effective, NIPT must be able to detect chromosomal rearrangements across the whole genome for a very low false-positive rate. Because standard NIPT can only detect the majority of larger (>6 Mb) chromosomal rearrangements and requires knowledge of fetal fraction, we consider that it is not yet ready for routine clinical implementation.

We examined six skeletons from mediaeval contexts from two sites in England for the presence of Mycobacterium leprae DNA, each of the skeletons displaying osteological indicators of leprosy. Polymerase chain reactions directed at the species-specific RLEP multicopy sequence produced positive results with three skeletons, these being among those with the clearest osteological signs of leprosy. Following in-solution hybridization capture, sufficient sequence reads were obtained to cover >70% of the M. leprae genomes from these three skeletons, with a mean read depth of 4–10×. Two skeletons from a mediaeval hospital in Chichester, UK, dating to the 14th–17th centuries AD, contained M. leprae strains of subtype 3I, which has previously been reported in mediaeval England. The third skeleton, from a churchyard cemetery at Raunds Furnells, UK, dating to the 10th to mid-12th centuries AD, carried subtype 3K, which has been recorded at 7th–13th century AD sites in Turkey, Hungary and Denmark, but not previously in Britain. We suggest that travellers to the Holy Land might have been responsible for the transmission of subtype 3K from southeast Europe to Britain.

Background The therapeutic landscape for mUC is rapidly evolving, with the introduction of immune checkpoint inhibitors (CPIs) and targeted therapies providing new options. Prognostic and predictive biomarkers are urgently needed to facilitate clinical decision-making. In this setting, ctDNA is a non-invasive method for genomic profiling, but its clinical utility remains largely untested. Methods Whole blood samples were collected for next-generation sequencing of leukocyte and cell-free DNA (cfDNA). Deep targeted sequencing was performed across a UC-specific custom 50-gene panel to a median unique read depth of 1040x for cfDNA. Clinical records were reviewed for baseline characteristics and outcomes. Results From 12/2014 until 11/2018, 103 patients with mUC underwent blood collection for cfDNA analysis. Of these, 84 had detectable ctDNA. Baseline characteristics and outcomes are presented in the table below for the entire cohort, along with exploratory analyses for patients who received platinum-based chemotherapy or a CPI in the metastatic setting. For cases with ctDNA>1%, selected results based on the presence or absence of mutations (muts.) in TP53 and ERCC2, as well as tumour mutation burden (TMB), are included. Table . 934P All cases (N = 103) Median age (range) 67 (37-88) Male (%) 83 Smoker (%) 63 Median ctDNA (%) 15.7 Median overall survival (OS, months) Present Absent P-value ctDNA > median 6.2 15.7 0.018 TP53 mut. 16.9 27.1 0.082 ERCC2 mut. 31.6 19.6 0.097 Platinum-treated (N = 54) Carboplatin (%) 37 First-line (%) 89 Median progression-free survival (PFS, months) Present Absent P-value TP53 mut. 6.0 6.7 0.76 ERCC2 mut. 13.4 5.5 0.028 CPI-treated (N = 50) Combination with CTLA-4 (%) 10 First-line (%) 54 Median PFS (months) Present Absent P-value TP53 mut. 3.3 3.2 0.92 ERCC2 mut. 2.9 3.3 0.22 TMB > 25 mut./Mb 4.4 2.9 0.67 Conclusions In our mUC cohort, the presence of ctDNA fraction above median correlated with worse OS, while mutations in ERCC2 correlated with improved PFS for platinum-treated patients. These data support the further exploration and validation of ctDNA as a tool for biomarker development in mUC. Additional analyses will be presented for the entire 50-gene panel. Legal entity responsible for the study The authors. Funding Canadian Institutes of Health Research (CIHR) Bladder Cancer Canada (BCC). Disclosure J. Lavoie: Honoraria (self): Astellas. N.L. Sundahl: Travel / Accommodation / Expenses: MSD; Travel / Accommodation / Expenses: Astellas; Travel / Accommodation / Expenses: Bayer; Travel / Accommodation / Expenses: BMS. D. Khalaf: Advisory / Consultancy: Bayer. T. Todenhofer: Advisory / Consultancy: MSD; Advisory / Consultancy: Roche; Advisory / Consultancy: BMS. C.K. Kollmannsberger: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Astellas; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy: Merck; Advisory / Consultancy, Travel / Accommodation / Expenses: Eisai; Honoraria (self), Advisory / Consultancy: Ipsen; Advisory / Consultancy: Janssen; Advisory / Consultancy: EMD Serono; Advisory / Consultancy: Roche. K.N. Chi: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Sanofi; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Janssen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Astellas; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy: Essa; Advisory / Consultancy: Amgen; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): Roche; Research grant / Funding (institution): Tokai Pharmaceuticals; Research grant / Funding (institution): Lilly/ImClone; Research grant / Funding (institution): BMS; Research grant / Funding (institution): Merck. B.J. Eigl: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Janssen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy: Zomanex; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Roche; Honoraria (self), Research grant / Funding (institution): Astellas; Honoraria (self), Research grant / Funding (institution): Pfizer. All other authors have declared no conflicts of interest.

Aim Accurate NGS-based HLA typing heavily depends on a reliable PCR amplification of HLA genes. Most protocols apply a method in which each HLA gene is amplified separately, after which amplicons are pooled and processed in a library preparation procedure. We have developed a reliable multiplex PCR for HLA-A, B, C, DRB1, DQB1 and DPB1, bringing down the number of amplifications from six to only one reaction per sample. Methods HLA gene-specific amplification primers, based on NGSgo-AmpX primers (GenDx), are combined in one PCR using the GenDx-LongMix mastermix. Amplicons are processed in the NGSgo® workflow (GenDx) for library preparation and sequenced on a MiSeq (Illumina) with application of paired-end sequencing (2x151 bp). Data is analysed in HLA typing software NGSengine®. Results A multiplex PCR was developed and optimized such that high quality NGS data was obtained and reliable HLA typings were obtained for a large validation panel n = >96). When comparing the data of the multiplex PCR data with the singleplex PCR, slightly elevated noise levels were observed due to formation of hybrid reads in Class I genes. With an optimized PCR protocol noise levels were reduced to acceptable levels. In addition, read depth difference between genes were minimized for efficient flow cell use. Conclusions A multiplex PCR for HLA-A, B, C, DRB1, DQB1 and DPB1 has been developed, that is fully compatible with the HLA typing workflow with NGSgo® for Illumina. PCR artefacts have been minimized with a new protocol that generates high quality data and supports reliable NGS-based HLA typing. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. M. van Heck: 5. Employee; Company/Organization; GenDx. K. van Ham: 5. Employee; Company/Organization; GenDx. M. Penning: 5. Employee; Company/Organization; GenDx.

Aim The GenDx NGSgo workflow for HLA typing using Next-generation sequencing (NGS) consists of four practical steps: amplification, library preparation, sequencing and data analysis. Out of these four steps the library preparation is the most labor intensive and most interspersed by incubations. To decrease hands-on time and reduce the risk of human error, NGSgo library preparation can be automated from start to finish using a liquid handler workstation. Here we describe the results of the automation of the NGSgo library preparation for Illumina on a Beckman Coulter Biomek 4000 workstation. The workflow was fully automated from amplicon pool to finalized library, making it completely hands-off to accommodate overnight runs and allow the operator to step away from the machine. Methods The Biomek setup included a thermal cycler, 96-well plate magnet, two peltiers devices for cooling 96-well plates and 1.5 ml tubes, a gripper tool, reagent reservoirs and liquid waste disposal. Pipetting was performed by two pipet tools. This setup allowed for flexible processing of 4 to 24 samples per run, with any number of loci. Per run only three boxes of pipetting tips were used for 24 samples. Panels with variable numbers of samples and replicate panels were processed with the Biomek. Subsequently, the pooled libraries were sequenced on a MiSeq system and the resulting data was analyzed with NGSengine software (GenDx). Results MiSeq run parameters were indicative of a high-quality sequencing runs. Read depth, mappability and noise levels of the sequence data presented here showed that the automated workflow resulted in high quality reads and accurate typings. Results from the replicate panel showed a 100% typing match. Conclusions – NGSgo library preparation for 4 to 24 samples can be performed on the Biomek 4000 platform, without manual intervention. – The automated workflow results in high-quality reproducible libraries. – Performing automated NGSgo library preparation as described here reduces hands-on time from 1.5 h to 10 min as compared to manual execution. B. Luiken: 5. Employee; Company/Organization; GenDx. E. Tavkin: 5. Employee; Company/Organization; Beckman Coulter. S. Parakh: 5. Employee; Company/Organization; Beckman Coulter. J. Lu: 5. Employee; Company/Organization; Beckman Coulter. A. Jackson: 5. Employee; Company/Organization; Beckman Coulter. S. van Wageningen: 5. Employee; Company/Organization; GenDx. M. Penning: 5. Employee; Company/Organization; GenDx.

Aim Current technologies for HLA sequencing-based typing often show ambiguous results, most of which are caused by cis–trans allele combinations. Next-generation sequencing (NGS) technology however generates sequences from single DNA molecules, meaning that paternal and maternal alleles can be uniquely identified. Therefore, NGS technology has great potential in the application of HLA typing for diagnostic purposes. The NGSgo ® workflow that proved to be compatible on the Ion PGM platform was now validated on the Ion S5 platform. Here we present NGS data generated when processing pooled HLA loci on the Ion S5 platform using the fully automated Ion Chef system. Methods Amplification of 96 clinical samples was performed for 11 HLA loci with NGSgo-AmpX that enables amplification of whole class I genes, and the essential genomic regions for high-resolution typing of the class II genes. The amplicons generated were pooled per sample and processed in a library preparation that consisted of three steps. The fragmentation and end-repair were performed with the NGSgo-LibrX in a single step followed by the ligation of universal and 24 unique barcoded adapters using the NGSgo-IndX kit (GenDx). Finally, after pooling the libraries in one tube, a magnetic bead step removed all fragments ® software (GenDx). Results Here we will present the data output showing the NGS data quality generated. A preliminary dataset already showed a locus mappability of 89%, read depth of 4206, ambiguities only for 3rd or 4th field resolution (except for DRB1 with 2 ambiguities in the 2nd field due to differences outside of the amplicon) and 100% concordance of the NGS typing with the available pre-types. Conclusions The availability of several NGS workflows allows the end-user to choose the most preferred HLA typing NGS platform that fulfills the requirements of their laboratory for their specific applications. Here we will demonstrate that the NGSgo workflow developed for the Ion PGM is also compatible with the Ion S5 platform, making it a powerful method for high-throughput HLA typings by means of NGS. E. Rozemuller: Stock Shareholder; Company/Organization; GenDx. N. Westerink: Employee; Company/Organization; GenDx. M. Penning: Employee; Company/Organization; GenDx.

Aim Genotyping of DRB1 and DRB3/4/5 is performed in our lab using generic DRB PCR primers, followed by sequencing on the 454 GS FLX and use of Conexio Genomics software. During genotyping homozygous cell lines, we detected additional sequences at about 7% of the read depth of the reported true allele. These artifacts appeared to be PCR crossovers between the DRB1 and DRB3/4/5 sequences. The crossovers usually had one or more mismatches with the IMGT database but occasionally matched a very rare allele (e.g. DRB1 ∗ 03:42). We performed a study to determine if these sequences were in vitro artifacts and to determine if some rare DRB alleles in the IMGT database might, in fact, be in vitro artifacts that had been submitted. Methods To test for in vitro artifact formation during late cycles of genomic PCR, we amplified the DRB loci from 21 homozygous cells lines under both our standard PCR conditions (35 cycles) and a reduced number of cycles (28), followed by 454 sequencing. Using the same PCR conditions, we also sequenced the DRB loci of four samples, each of which had previously been identified as having a novel DRB allele that was subsequently submitted to the IMGT database. Results For all homozygous 21 cell lines, amplification for 35 cycles gave low abundance sequences as possible “second alleles.” In 36% of the cases, these artifactual sequences corresponded to named alleles. Such sequences were not detected with amplification for 28 cycles. In the case of 4 samples with rare alleles previously submitted to the IMGT, we found that 3 were true alleles. The fourth, while not a crossover product, contained a sequencing error which was revealed by our 454 HLA genotyping assay. Conclusions Formation of in vitro crossover products of the DRB loci can occur in late cycles of PCR. PCR conditions have been identified that minimize generation of these in vitro artifacts. Clonal sequencing on the 454 GS FLX is a valuable method for revealing both these artifacts and traditional sequencing errors.