scope:

This guideline specifies recommendations for interpreting and reporting results of Sanger-based (dideoxynucleotide chain termination) sequencing of broad-range DNA targets for identifying pure isolates of bacteria, mycobacteria, and fungi from cultured patient isolates. Partial- and full-gene sequencing with 16S ribosomal RNA (rRNA) genes for bacterial and mycobacterial identification, as well as internal transcribed spacer (ITS) regions (ie, ITS-1 and ITS-2) for fungal identification are covered, including alternative DNA targets when appropriate. Although massively parallel (next-generation) sequencing technologies are rapidly emerging, this guideline's scope is limited to Sanger-based targeted DNA sequencing.

To assist the medical laboratory, guidance is provided for:

• Selecting DNA targets and sizes for amplification and sequencing

• Establishing QC parameters for amplification and sequencing

• Measuring sequence quality

• Assessing reference sequences and databases

• Comparing sequences for identification

• Establishing interpretive criteria for identity scores generated by targeted DNA sequencing

• Developing clinically relevant reporting strategies for specific microorganism groups

• Identifying the limitations of targeted DNA sequencing for microbial identification

The intended users of this guideline are medical laboratories and laboratories performing amplification and Sanger-based (dideoxynucleotide chain termination) sequencing of broad-range DNA targets for identifying bacteria, mycobacteria, and fungi from cultured patient isolates.

This guideline does not:

• Include procedures for performing microbial sequencing.

• Include RNA targets for sequencing.

• Provide guidance on definitive taxonomical criteria for microorganism classification or identification methods for novel microorganisms.

• Cover alternative sequencing systems or specific molecular assays designed with these broad-range DNA targets.

• Discuss typing strains for epidemiological purposes.

• Discuss virus or parasite identification.

• Discuss amplification and sequencing directly from patient specimens.