A Non-Destructive High-Speed Procedure to Obtain DNA Barcodes from Soft-Bodied Insect Samples with a Focus on the Dipteran Section of Schizophora

Abstract

While the need for biodiversity research is growing, paradoxically, global taxonomical expertise is decreasing as a result of the neglected funding for young academics in taxonomy. Non-destructive approaches for DNA barcoding are necessary for a more efficient use of this dwindling expertise to fill gaps, and identify incorrect entries in sequence databases like BOLD or GenBank. They are efficient because morphological re-examination of species vouchers is still possible post-DNA barcoding. Non-destructive approaches for Diptera with a comprehensive species representation or the consideration of diagnostic fragile morphological characters are missing. Additionally, most non-destructive approaches combine a time intensive and non-destructive digestion step with common DNA extraction methods, such as commercial kits or CTAB DNA isolation. We circumvented those approaches and combined a modified non-destructive TE buffer high-speed DNA extraction, with a PCR inhibitor-resistant PCR reaction system, to a non-destructive DNA barcoding procedure for fresh and frozen samples of the Schizophora (Diptera). This method avoids morphological impairment and the application of harmful chemicals, is cost and time effective, restricts the need for laboratory equipment to a minimum, and prevents cross-contamination risk during DNA isolation. Moreover, the study indicates that the presented non-destructive DNA barcoding procedure is transferable to other soft-bodied insects. We suggest that PCR inhibitor-resistant master mixes enable the development of new—and the modification of existing—non-destructive approaches with the avoidance of further DNA template cleaning.

Identifying the best PCR enzyme for library amplification in NGS

“…As a result we would expect these enzymes to perform well on other genomes irrespective of complexity and GC content, especially when using pure genomic DNA as used here. It should be noted that RepliQa is also reported to be tolerant to many known PCR inhibitors that can be present in crude extracts and has been shown to efficiently amplify targets from soil [ 27 ] and insects [ 28 ]. Given that these enzymes give efficient amplification of both high and low GC content loci, though not tested here, it is expected that they would be particularly suited for amplification of metagenomes and microbial communities containing many different species at different percentages and with widely varying GC content genomes.…”

Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar

“…Additionally, there are numerous cellular constituents that can act as DNA polymerase inhibitors including proteins, polysaccharides, cell debris and exogenous DNA [ 38 ]. Further work would be required to optimise this reaction or trial alternative enzymes that are resistant to PCR inhibition such as those recently reported by Stein and co-workers [ 39 ]. Alternatively, reducing the EDTA concentration in Buffer 1 could also be trialled, although this is already relatively low and may affect the long-term preservation of isolated DNA in crude preparations.…”

Identifying the best PCR enzyme for library amplification in NGS

“…As a result we would expect these enzymes to perform well on other genomes irrespective of complexity and GC content, especially when using pure genomic DNA as used here. It should be noted that RepliQa is also reported to be tolerant to many known PCR inhibitors that can be present in crude extracts and has been shown to efficiently amplify targets from soil [21] and insects [22].…”