Table 2 Summary of different technologies for molecular identification of fish genetic sex.

Method	Time^a	Estimated cost^b	Required operational skills	Instruments required	Advantages	Disadvantages
PCR + electrophoresis	~ 4–6 h	$ 0.05 ~ 0.1	Regular molecular biology skills	Regular thermal cycler Gel imaging system	Low cost	Need to be performed in a regular molecular biology lab Time consuming
PCR + Sanger sequencing	~ 1 day	$ 3 ~ 8	Regular molecular biology skills Specialized skill for operating Sanger sequencer	Sanger sequencer, or Genetic Analyzer	Accuracy	High cost per sample Need to be performed in a molecular biology lab with an expensive Sanger sequencer^c Time consuming
RAA-CE	~ 30 min	$ 6.2^d	Regular molecular biology skills Specialized skill for operating capillary electrophoresis	Dry bath with 96-well block	Time saving	High cost per sample Need to be performed in a molecular biology lab with an expensive CE
RAA-LFD	~ 30 min	$ 6.2^d	Regular molecular biology skills	Dry bath with 96-well block	Time saving Convenience Potentially versatile testing scenarios	High cost per sample

^aEstimated turnaround time of the sex identification methods. The time for reaction preparation, reaction and result visualization included, while the time for nucleotide extraction not included;
^bCost per reaction was estimated based on the prices of consumables and reagents in Anhui Province, China. Cost may vary depending on the sources of consumables and/or reagents;
^cOutsourced Sanger sequencing usually lead to longer turnaround time (2–5 days);
^dRAA-CE cost breakdown: the cost for RAA kit is $3.7 per sample; the cost for CE is $1.8 per sample; the cost for regular PCR reagent is $0.1 per sample; the cost for a set of labeled primer/probe is ~ $0.6 per sample. RAA-LFD cost breakdown: the cost for RAA kit is $3.7 per sample; the cost for LFD is $1.8 per sample; the cost for regular PCR reagent is $0.1 per sample; the cost for a set of labeled primer/probe is ~ $0.6 per sample.

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