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I have been in sequencing of DNA barcode region in insect species. Direct sequencing of amplified DNA often results in misreading of first 15-25 nucleotides on the 5' end, resulting in shortened sequences. If sequencing fails the procedure requires reamplification which is not possible partcularly with difficult samplees such as museum stored dried insect samples. Direct sequencing also requires use of barcode primer(s) as sequencing primers. Although a successful sequencing results in decreased cost, as i have seen PCR cloning of amplified fragments and sequencing with universal M13 primers provides advantages such as i) sequence for complete barcode region, ii) permanant availability of amplified fragment for storage and reutilization and iii) consistency in sequencing procedure using M13 primer(s) irrespective of the primers used for amplifying DNA barcode regions.

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