scope:

This guideline specifies recommendations for clinical laboratories that employ amplification and Sangerbased (dideoxy-termination) sequencing of broad-range DNA targets for the identification of bacteria, mycobacteria, and fungi from cultured clinical isolates. Partial and full gene sequencing with 16S rRNA gene for identification of bacteria and mycobacteria, and internal transcribed spacer regions ITS1 and ITS2 regions for identification of fungi, are addressed with inclusion of alternative DNA targets when appropriate. To assist the clinical laboratory, this document provides guidelines for:

selection of DNA targets and size of targets for amplification and sequencing;

establishment of quality control parameters for amplification and sequencing;

measurement of quality of sequence;

assessment of reference sequences and databases;

comparison of sequences for identification;

establishment of interpretive criteria for identity scores generated by gene sequencing;

reporting strategies that are clinically relevant for specific groups of microorganisms; and

limitations of gene sequencing for microbial identification.

This guideline is not intended to:

address RNA targets for sequencing;

provide guidelines for definitive taxonomical criteria for classification of microorganisms or methods to identify novel microorganisms;

address alternative sequencing systems or specific molecular assays designed with these broad-range DNA targets;

type strains for epidemiological purposes;

identify viruses or parasites; or

address amplification and sequencing from direct specimens.