Table A.2.

Upstream analytical process accreditation requirements as published by CAP's NGS Work Group MOL (Aziz et al., 2015). The table summarizes seven laboratory checklist requirements for the wet bench process in NGS experiments.

MOL topic	Description	Requirements
Documentation	Use of SOP	All standard operating protocols must be documented in order to trace each step and manipulations All used methods, reagents, instruments, instrument software and versions have to be documented Controls need to be described Targeted NGS assays and target-enrichment protocols, regarding captured regions must be documented Development of SOP for each validated sample type Sequence information of barcodes for pooled analysis must be documented Metrics and QC parameters regarding run performance have to be documented (% of reads mapping to target region, base quality and coverage thresholds, average coverage/base and target region) Definition and documentation of acceptance and rejection criteria for sample preparation and sequencing Documentation of regions that failed analysis Written SOP for evidence of compliance
Validation	Validation and revalidation of processes after establishment of modifications	Analytic performance of NGS procedures must be validated Determination of optimal assay conditions and analysis settings in order to optimize each workflow step Determination of performance parameters (sensitivity, specificity, accuracy, reproducibility, reliability, detection limit) Validation of each sample type Determination of a minimum number of samples, dependent on sample, target, assay and application Revalidation of minor changes such as new reagent charges Documentation of lot numbers Revalidation of major changes such as new sequencing system or different target enrichment method
QM	Use of documented QM program	Development of a QM plan QA program must be written and compliance has to be documented QA program should assess all parts of a workflow QA program should be embedded within overall QMS Quality program should address general problems, especially in case of nonconformance with laboratories own policies and procedures Quality program should encourage scientific and laboratory staff to interchange about quality issues
Confirmatory testing	Use of policy for documentation of confirmatory testing	Established policy that clearly documents indications for confirmatory testing Each laboratory has to show evidence of monitoring their NGS assays
Laboratory records	Use of laboratory records for identification and trace of samples	Documentation of all reagents, primers, sequencing chemistries and platforms Description of performed test comprising targeted sequence and depth of coverage Citing publications or websites Maintaining a documentation system
Exception log	Use of exception log for monitoring deviations from SOP	Documentation of any deviation from SOP, the reason for deviation and the outcome Extensive consultation with laboratory supervisor or laboratory director regarding testing issues Documentation of troubleshooting, resolution and appropriate communications Changes or modifications in SOP should be supported by two qualified staff members and have to be signed by the laboratory director
Monitoring of upgrades	Use of policy for monitoring, implementing and documentation of upgrades	Implementation of policy to monitor and integrate upgrades to instruments, sequencing chemistries and reagents/kits Validation of introduced up-to-date instruments or reagents/kits Addressing methods which are used to monitor upgrades