Literature review (2000)
Discuss the key methods of species detection and identification and how/why new emerging molecular techniques are beginning to replace some of these approaches. You should describe the benefits and drawbacks of these new approaches and provide examples to support your answer throughout.
Introduction
With poaching and rapid decline of protected species due to illegal trade of rare goods such as ivory, wildlife law enforcement is in need of much development. Wildlife DNA forensic is a field receiving increasing attention due to its admissibility in court (Ogden, Dawnay and McEwing, 2009). Species identification may be used in these cases such as poaching (Gupta, Verma and Singh, 2005) and trade of endangered/protected species, commodities such as shark fins (Shivji et al., 2002). This review will compare methods of species detection and look at the strengths and limitations of existing and new emerging techniques.
Isolation and analysis of variations between individual species gene markers are the key factors of DNA forensics. The particular area of interest is Mitochondrial DNA (MtDNA) as they exist in greater number per cell than nuclear DNA, and can exist in trace samples due to its circular form and protective mitochondrial casing. MtDNA is useful for species detection as they are, aside for mutations, directly maternally linked (HUTCHISON et al., 1974). These samples can be compared with known relative samples. The key regions of interest of MtDNA for species identification are the coding regions cytochrome b (cytb) and cytochrome c oxidase unit I (COI). Cytb was the traditional gene used for species analysis until 2003 when the use of COI was introduced called barcoding. A debate into which region offers high inter- species variability and low intra-species variation to accurately identify species. Tobe, Kitchener and Linacre (2009) set out to settle the debate using cytb and COI sequences from 236 samples and found that the use of the cytochrome b gene will offer greater informative value in a smaller fragment however, there was little difference in intra species variation between the use of the two regions.
DNA sequencing
Once MtDNA is extracted it is subject to polymerase chain reaction (PCR), there are varying methods of amplification using either universal primers or species specific. Primer choice largely depends on sample size. Universal PCR primers enable amplification of DNA from a wide range of species without any prior information regarding the sample, this is shown by Verma & Singh (2003) who of a sample of 221 animal species could reveal the source though simply using universal primers to amplify a specific segment of the cytb region of MtDNA. However, a drawback of the use of universal primers is that when amplifying DNA from a mixed samples a universal primer would amplify all/most of the present DNA, therefore sequence results would be messy presenting difficulty during analysis.
Following amplification via PCR the sample can be analysed in a few different ways. Sequencing and analysis of sequence data of mitochondrial DNA is one of the most widely used methods of species detection (Carracedo et al., 2000). Sanger sequencing is the most common validated technique however there are new emerging technologies for sequencing. Mass parallel sequencing, also termed Next Generation Sequencing (NGS) or high-throughput sequencing, is massively parallel sequencing millions of fragments in a single run versus sanger sequencing which only produces one forward and reverse read (Gurson, 2018) NGS has a nigh sensitivity to low frequency variants which aids detection of population differentiation through identification of single nucleotide polymorphisms (SNP) as shown in a study by McCormack et al (2012) who detected population differentiation in four bird systems Using single nucleotide polymorphisms (SNPs) mined from the loci.
Ogden, R. (2011). Describes the pros and cons of various methods of high-throughput sequencing methods, firstly Reduced representation libraries (RRL) are created by restricting whole genomic DNA with a simple restriction enzyme, then sequencing only a size specific fraction of the restricted product product (e.g. 200 bp fragments) the complexity to which the genome is reduced is dependant of the GC content, genome size and choice of enzyme. The fragment lengths are observed through electrophoresis . This method formed the basis of SNP discovery in cattle as shown my (Van Tassell et al. 2008) who identified 62,042 putative SNPs and predicted their allele frequencies from the DNA of 66 cattle. This method (RRL) however requires many sequence runs to cover the whole genomic sequence and is very high cost.
cDna sequencing constructs a library of sequences of the transcriptome of RNA. this approach increases the proportion of coding DNA available for sequencing yet the power of cDNA libraries to reduce genome complexity remains difficult to quantify. The drawbacks of this method is that the process is complicated by intron–exon boundaries and costing of the method is high. Ogden, R. (2011).
AD-Tag sequencing. RAD-Tag sequencing (Miller et al. 2007; Baird et al. 2008) combines restriction endonuclease reduction of genome with targeted NGS, the process allows individuals (or groups of individuals) to be labelled and identified within a single sequencing lane. Sequence depth is calculated differently to the RRL method whereby the number of loci (or RAD-Tags) sequenced are predicted and therefore sequence depth is more accurately predicted. One disadvantage of RAD-Tag sequencing for SNP discovery is that in the absence of a reference genome sequence, the flanking region of SNPs in the forward read will often be too short to design genotyping assays for any SNPs observed Ogden, R. (2011).
Sequence outputs are compared with known samples on online reference databases such as NCBI GenBank. (Ncbi.nlm.nih.gov, 2018). GenBank has been/can be incredibly useful due to its vast storage for sequence data for sample reference/comparison. However, it must be taken into account that this software has limited regulation as to who sourced/collected the data. Some sequences on the database may be inaccurate. As shown by Seah, Ariffin & Jaafar (2017) who found when searching reference databases that of 232 downloaded sequences,88 sequences were detected as potential misidentification as these sequences did not group with their own taxa. Concluding that the accuracy of deposited sequences should be monitored to ensure success of species identification. Results from this show that an analyst must exercise caution when using GenBank derived data and perhaps test the sequence match by confirmatory testing a sample of the matched species.
Johnson, Wilson-Wilde and Linacre, (2014) sate that the collection of data is only of value if it can be interpreted, evaluated and supported conclusions made. Following Sequence matching from various technological software such as Molecular Evolutionary Genetics Analysis (MEGA) to analyse sequence data. a phylogenetic tree is frequently constructed. The nodal length and bootstrap support will indicate the relationships and strength of the population group and the unknown sample. Currently there is little international standardisation as to which program to use; this has potential problems when presented in the criminal justice system.
Single nucleotide polymorphism (SNP)
SNP typing or genotyping testing is faster and cheaper as the test does not require development of such long fragments of high quality DNA, however less information is gained in comparison to DNA sequencing (Ogden, Dawnay and McEwing, 2009). An advantage of SNP testing is particularly useful in cases of traditional medicines where there may be numerous species in the same concoction as current DNA sequencing technology cannot differentiate between mixtures. (Tobe & Linacre 2010). There are multiple methods for typing SNP markers.
sNapshot single nucleotide polymorphism
Double stranded DNA is heated to denature the two strands. A primer is introduced that is the compliment and ends one base 3’of the targeted SNP. The DNA polymerase can only add dideoxy modified bases, this modified base terminates the chain by preventing further binding down the strand. The presence of a fluorescent marker indicates the presence of the targeted SNP (Tobe & Linacre 2010). Kitpipit, et al (2012). Developed a single SNaPshot multiplex assay to detect SNPs in species of tiger and method was found to be reliable, accurate, specific, sensitive and robust.
A second version of SNP testing is to make use of the SNP variations that are species specific to design species-specific primers. The use of species specific primers would only amplify the DNA of the specific species chosen (if present in the sample). Therefore useful in situations whereby samples contain a mixture of species DNA (Tobe & Linacre 2010). However, the main disadvantage of this technique is that the test has to incorporate all the potential species that are most likely to be present. If the correct DNA primer is not found for the unknown sample there will be no detection. Therefore, this test can only be useful if there is a defined group of species suspected in the sample.
PCR-RFLP
Restriction fragment length polymorphism (RFLP) is another method of species detection, following the same steps of extraction and amplification, the sample is subjected to PCR with a species-specific restriction endonucleases that cut the DNA at a specific coding region.
The restriction endonucleases cut positions vary between species due to where the binding site appears on the genome.
An enzyme is selected depending on how different the cut positions are between species to leave significantly different fragment lengths. When the amplified sample is subject to Agarose Gel Electrophoresis (AGE) the amplified fragment lengths are compared to the cut positions for the suspect species. A study by Zehner, Zimmermann & Mebs (1998) used PCR-RFLP to distinguish between human and several animal species from samples of roasted meat, stomach contents and bone. The single restriction endonuclease ALU I was able to differentiate between species even in mixed samples, however had no cut positions for chicken or turkey.
Microsatellite genotyping
Microsatellite markers show differences between sequences of DNA through varying numbers of repeated base sequences. The variant causes a change in fragment length which can be observed under Gel Electrophoresis. A case example of the use of microsatellite genotyping for tracing back the identity of a tiger of which a claw (the sample) was found. Seven tiger-specific microsatellites were used to identify whether the seized claw was that of a massacred tigress from a zoo park in India.( Gupta, S.K. et al. 2011)
Conclusion
Sequencying results are highly validated for use in forensic cases and
Both PCR-RFLP and SNP-genotyping methods are widely accepted within the forensic genetic com- munity, however they are applied in the context of species detection, rather than species identification.
References
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