Metadata Annotation Guidelines¶

Purpose of this Document¶

This document describes the RECOMMENDED ways to provide metadata annotation for various experimental setups.

Clarification of Terms¶

The key words “MUST”, “MUST NOT”, “REQUIRED”, “SHOULD”, “SHOULD NOT”, “RECOMMENDED”, “MAY”, and “OPTIONAL” in this document are to be interpreted as described in [RFC2119].

Individual fields¶

library_generation_method¶

The library_generation_method describes how the nucleic acid annotated in template_class that encodes the V(D)J-rearrangement it reverse-transcribed, amplified and/or otherwise prepared for further processing. Typically this procedure will precede further NGS platform- specific steps, however these procedures MAY be combined. The field uses a controlled vocabulary, the individual values are described below:

`template_class`	`library_generation_method`	Methodology
`DNA`	`PCR`	Conventional PCR on genomic DNA of a vertebrate host (requires: `synthetic` == `false` )
`DNA`	`PCR`	Conventional PCR on DNA of a synthetic library (requires: `synthetic` == `true` )
`RNA`	`RT(RHP)+PCR`	RT-PCR using random hexamer primers
	`RT(oligo-dT)+PCR`	RT-PCR using oligo-dT primers
	`RT(oligo-dT)+TS+PCR`	5’-RACE PCR (i.e. RT is followed by a template switch (TS) step) using oligo-dT primers
	`RT(oligo-dT)+TS(UMI)+PCR`	5’-RACE PCR using oligo-dT primers and template switch primers containing unique molecular identifiers (UMI), i.e., the 5’ end is UMI-coded
	`RT(specific)+PCR`	RT-PCR using transcript-specific primers
	`RT(specific)+TS+PCR`	5’-RACE PCR using transcript- specific primers
	`RT(specific)+TS(UMI)+PCR`	5’-RACE PCR using transcript- specific primers and template switch primers containing UMIs
	`RT(specific+UMI)+PCR`	RT-PCR using transcript-specific primers containing UMIs (i.e., the 3’ end is UMI-coded)
	`RT(specific+UMI)+TS+PCR`	5’-RACE PCR using transcript- specific primers containing UMIs (i.e., the 3’ end is UMI-coded)
	`RT(specific)+TS`	RT-based generation of dsDNA without subsequent PCR. This is used by RNA-seq kits.
any	`other`	Any methodology not covered above

Specific Use Cases and Experimental Setups¶

Synthetic libraries¶

In synthetic libraries (e.g. phage or yeast display), particles present genetically engineered constructs (e.g. scFv fusion receptors) on their surface. As this deviates substantially from other workflows, the following annotation SHOULD/MUST be used:

In general, Subject should be interpreted as the initial library that undergoes a mutation/selection procedure.
synthetic: MUST be set to true
species: It is assumed that every synthetic library is derived from V and J genes that exist in some vertebrate species. This field SHOULD encode this species. Importantly, it MUST NOT encode the phage vector, the bacterial host or the comparable biological component of the library system that constitutes the presenting particle.
sample_type: SHOULD be NULL.
single_cell: Only true if individual particles are isolated and sequenced. Note that colonies or plaques, even if containing genetically identical particles, per se do not match this definition and therefore MUST be annotated as false.
cell_storage: SHOULD be used for non-cellular particles analogously.
physical_linkage: For scFv constructs the hetero_prelinkeded term MUST be used. VHH (i.e. camelid) libraries SHOULD annotate none as there is only a single rearrangement envolved.

AIRR Standards 1.3 documentation