Munich Leukemia Laboratory (MLL), located in Munich, Germany, works with hospitals and hematologists, as well as with pharmaceutical companies, from all over the world to provide diagnoses and prognoses for leukemia and lymphoma patients using cytomorphology, cytogenetics, FISH, and molecular genetics. Since the company’s beginning in 2005, they have grown from 29 to 199 employees and from processing 3700 samples per year to more than 91,000 samples per year (Figure 1A). A large number of cases are investigated by next generation sequencing (NGS) using MLL myeloid and lymphoid panels manufactured by IDT and IDT’s xGen Exome Research Panel, as well as amplicon-based assays (MPN, single amplicons) (Figure 1B). Together, IDT and MLL are shaping the future of hematological diagnostics.
We spoke with one of the company’s founders, Prof Dr Dr Torsten Haferlach, an expert in hematology and oncology, about the methods MLL uses to decrease turnaround time, increase efficiency, and provide scalable solutions.
Figure 1. IDT’s xGen panels have supported MLL’s progress in increasing the number of samples processed via next generation sequencing (NGS). (A) Number of samples processed per year at MLL. (B) Numbers of samples analyzed by NGS. Three different assays are given: MPN (small amplicon-based panel), Panel (myeloid and lymphoid panels), and SA (single amplicon). The numbers for 2019 are extrapolated based on the first 6 months of the year. (Figure courtesy of Munich Leukemia Laboratory.)
NGS improves diagnostics
MLL was an early adopter of NGS technology. It was the first company in Europe to be accredited according to DIN EN ISO 15189 and has been able to use NGS to build an extensive mutation database. “I can’t even think about a time where we didn’t use NGS for molecular investigations,” says Dr Haferlach. Additionally, because MLL receives samples for testing from all over the world, the company has acquired an equally extensive sample database. Their extended history of using NGS for diagnostic purposes puts the company in a unique position to optimize technologies that others are only just beginning to utilize. The company continues to improve their accredited diagnostic reports with the information gained from new genes that are being investigated. Workflows, turnaround time, and database storage are all fine-tuned to give the best prognostic information and recommendations for precision medicine.
MLL provides clinicians a report that lists all the analyzed genes in a panel. For each gene, called variants are listed along with coverage, resulting sensitivity, and a short explanation of the prognostic or diagnostic impact on the disease.
Figure 2. MLL’s next generation sequencing (NGS) workflow. (Figure courtesy of Munich Leukemia Laboratory.)
Decreasing turnaround time
Turnaround time is less than a week, from receiving the samples to delivering the diagnostic report (Figure 2). Dr Haferlach states that using xGen panels has been instrumental in allowing MLL to decrease turnaround time. When the company requires probes targeting specific genes for any clinical or customized study, MLL can order a custom panel from IDT. The probes are delivered before the first sample arrives, with no added time to the diagnostic workflow.
Another critical requirement for fast turnaround time is their ability to use automation. MLL uses two different Illumina library preparations, pooling them together for the IDT enrichment protocol. These workflows are automated on a liquid handling robot. IDT’s enrichment protocol provides the flexibility of use with a variety of preparation protocols, a quality that is unique to IDT. This flexibility is important to MLL because the company analyzes multiple sample types, including formalin-fixed paraffin-embedded (FFPE) material and liquid biopsies.
Selecting capture technology
MLL needs 100s of unique dual indexes (UDI) to leverage the throughput of NovaSeq™, which IDT can accommodate. With our custom adapters, MLL can now load up to 384 samples on the flow cell, depending on the library size per sample, as compared to 96 previously. This exploits the sequencing capacities and minimizes the number of runs.
When comparing capture technologies, MLL scientists were particularly impressed with the uniformity of IDT’s enrichment-based panels. Capture uniformity, or the median coverage required to cover a region of interest, is key for MLL’s projects. If a region does not reach 400X coverage, the respective region must be rescheduled and processed in a subsequent sequencing run, increasing the time required to deliver a report. The uniformity of IDT’s enrichment panels allows most captures to be covered in a single run.
MLL recently switched over to the NovaSeq 6000 instrument in the routine diagnostic workflows, using the NovaSeq system with the S1 flow cell, dramatically increasing the number of reads per sequencing run. This also increases the data output, as the NovaSeq instrument produces up to 400 GB on a S1 flow cell and 3 TB on a S4 flow cell per sequencing run. After sequencing, analysis is run using the BaseSpace Sequence Hub™, Illumina’s cloud-based computing environment. The scientists use apps in the BaseSpace Sequence Hub to demultiplex, align, and call variants. Analyses with the BaseSpace apps are performed on routine diagnostics, such as the myeloid and the lymphoid panels, which are small panels composed of fewer than 100 genes. Currently, overall analysis time takes around 30 minutes regardless of the number of samples per run. This fast runtime is supported by cloud computing, because samples are analyzed in parallel, allowing unlimited scaling to keep runtime low. Previous analyses were run serially, which leads to linear compute time, so each sample for each patient took 20 minutes to analyze. With linear computing, the more samples there are, the longer it takes to compute the data. Cloud-based computing decreases overall computing time.
Customization without sacrificing quality
In addition to the routine diagnostics of leukemia and lymphoma patients, MLL provides both custom panels for specific pharmaceutical research studies and established panels that can be used by multiple companies or scientists for a variety of research studies. Importantly, MLL is accredited, so every assay must be accredited. MLL relies on the flexibility of IDT’s products for clinical diagnostics, as well as the quality to maintain their accreditation status. Any flexibility options must be in line with the quality expected from an accredited company.
Further, MLL now implements whole genome sequencing (WGS) as well as whole transcriptome sequencing (WTS). These studies are validated using orthogonal data. Dr Manja Meggendorfer, head of MLL’s molecular genetics department, and head of R&D, says that while the interpretation of the data is difficult, they have found that whole genome sequencing confirms the cytogenetic data as well as panel and exome sequencing results. MLL is now performing orthogonal testing in more than 4500 patients with WGS and WTS to determine if copy number variations (CNV) and structural variants (SV) can be identified with WGS and WTS, replacing the gold standard techniques of today. IDT’s xGen CNV Backbone Panel
was designed to provide an even representation of the genome, including single nucleotide polymorphisms (SNPs), to enable copy number calling. MLL is testing this panel, now incorporated in their NGS panel workflow, for use of CNV analyses, aiming to eliminate the need for separate chromosomal banding analyses in the future.
IDT is helping MLL change the game for clinical diagnostics labs by utilizing NGS for laboratory-developed tests. Scalability, flexibility, customization, and automation all work together to maximize quality and turnaround time. NGS provides genomic data to inform clinical decisions and improve patients’ health.