Project Meeting
After a first contact with the BSF, ideally via bsf@cemm.at, an initial meeting may help accelerating the intended project. We provide consulting already as early as the study planning stage, but we assume that the intended research project has been positively reviewed by an ethics committee. We can assist in choosing the optimal NGS assay, the required number of samples and replicates and how to optimise the sampling to get the highest possible data quality. Ideally, our clients can find out about the feasibility of the project, its time frame, the required budget and an initial analysis plan. We can also provide quotes for grant applications. In our experience both, on-line or in-person meetings work. Generally, the BSF has built capacity to accommodate also larger projects without prior waiting times.
Project Initiation
The BSF requires metadata documentation for both, the project-level and for each submitted sample. Rich and meaningful metadata annotation facilitates our initial analyses without the requirement for further communication at the time of analysis. The FAIR principles of data management – Findable, Accessible, Interoperable, and Reusable – greatly aid raw data submissions to public sequence archives such as the EMBL-EBI Array Express or the NCBI GEO. We also need a Sequencing Authorisation Form signed by the budget holder, usually the principal investigator.
Sample Submission
After submitting the project’s metadata annotation and performing the experiment, samples can be submitted to the BSF. We generally start from extracted DNA or RNA, small pools of sorted cells in lysis buffer or fixed cell suspensions. Please ensure that especially RNA material arrives on dry ice. Since our NGS Technologists perform lengthy and complex NGS protocols with few safe stopping points we can only accept in-person submissions on Wednesdays between 15:00 and 17:00 and on Fridays between 13:00 and 15:00.
Submissions of live single cell suspensions need to be announced well in advance to ensure that all reagents are on stock and NGS technologists are available at the time to process the samples immediately. We will provide appointments on top of the regular submission times.
Sample material submitted via mail should be sent early in a week to avoid shipping interruptions during weekends.
Initial Quality Checking
The BSF critically scrutinises all input material to optimise the outcome of the rather expensive NGS assays. We will report back in case any sample fails our quality thresholds and potentially ask for a resubmission. Of course, sample material can be scarce and precious so that sometimes, compromises must be made. In such cases we will always inform you and confirm with you before beginning any costly sample preparation procedures.
Library Preparation
In case a library preparation service was requested, we aim for optimising both, economy, by bundling with other customer projects, and, turnaround time, by processing urgent samples as rapidly as possible. The library preparation can still take a bit of time because NGS protocols can be complex and the BSF performs quality checks at critical intermediate steps.
Sequencing Service
Ready-made and in-house prepared libraries undergo rigorous quality checks and can sometimes be pooled with others, again under economic aspects, optimising the achievable output. The BSF monitors sequencing run performance closely and releases data only after confirming its quality requirements. In the rare event of a sequencing run failing due to instrument errors or vendor chemistry errors it will be rerun automatically. However, customer-provided NGS libraries that fall outside the supported criteria, with regards to custom sequencing primers, unusual sample index sequences, low-complexity payload sequences, NGS adapter dimers or unusual size profiles and which ultimately fail sequencing cannot be rerun and will be charged according to the costs incurred. Since the BSF applies rigorous quality checks of submitted NGS libraries, most issues will be noticed early and reported already before the sequencing.
Raw Data Conversion and Initial Analysis
Raw sequencing run data is converted into standard NGS formats of sequence and quality information and made available via a secure web server. If requested, an initial data analysis can be performed by the BSF, and the results are also made available via a secure web server. The analysis usually involves alignment to reference genome assemblies, differential expression, methylation, peak or genotype calling. Further quality control steps beyond sequence performance, and the overall performance of the experiment is reported in an e-mail. The BSF utilises the powerful CeMM compute cluster so that the analysis of sensitive data is performed in-house.
Project Conclusion
Further consulting regarding the project’s outcome is available. If desired, further meetings can be held to discuss the results, the analysis steps or a follow-on project.
Data Handover and Archiving
Although the BSF writes both, sequencing runs and the extracted raw data onto magnetic tapes, a restore after a longer period could still fail. It is thus our client’s responsibility to archive the raw data on their side. Initial analyses provided by the BSF are generally not stored for long term and may not be entirely reproducible with newer and improved software at a later stage. It is thus a good idea to also archive the initial analyses on your side. The BSF is more than happy aiding in the submission to public archives for NGS data, such as the EMBL-EBI Array Express and the NCBI Gene Expression Omnibus (GEO) for non-personally identifiable data or the European Genome Phenome Archive (EGA) for personally identifiable data sets.
Invoicing
After completing a sequencing request, we will send advance billing information to the contact person before submitting an invoice to the billing contact. We can also handle urgent invoicing requests to meet project account closing deadlines. Importantly, in line with the law, we can only issue an invoice after completing the service. However, we can issue advance invoices which will be labelled as such but may not be accepted by all funding agencies. Please inquire at your project funding agency.
Acknowledgement
To maintain the service long-term, the BSF needs to convince funders of its utility and importance. We can best do that if you acknowledge our contribution in your scientific publications.
Next-generation sequencing [and an initial data analysis] was performed by the Biomedical Sequencing Facility at CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences.
For projects under a commercial fee-for-service arrangement, an acknowledgment is not strictly required but would be still nice to have.
