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Selective enrichment and sequencing of whole mitochondrial genomes in the presence of nuclear encoded mitochondrial pseudogenes (numts).

Wolff JN, Shearman DC, Brooks RC, Ballard JW - PLoS ONE (2012)

Bottom Line: We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts.Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence.This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia. jonciwolff@yahoo.com

ABSTRACT
Numts are an integral component of many eukaryote genomes offering a snapshot of the evolutionary process that led from the incorporation of an α-proteobacterium into a larger eukaryotic cell some 1.8 billion years ago. Although numt sequence can be harnessed as molecular marker, these sequences often remain unidentified and are mistaken for genuine mtDNA leading to erroneous interpretation of mtDNA data sets. It is therefore indispensable that during the process of amplifying and sequencing mitochondrial genes, preventive measures are taken to ensure the exclusion of numts to guarantee the recovery of genuine mtDNA. This applies to mtDNA analyses in general but especially to studies where mtDNAs are sequenced de novo as the launch pad for subsequent mtDNA-based research. By using a combination of dilution series and nested rolling circle amplification (RCA), we present a novel strategy to selectively amplify mtDNA and exclude the amplification of numt sequence. We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts. Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence. This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error.

Show MeSH
Test for the presence of mitochondrial and nuclear DNA in serial dilution series' T-DNA I & T-DNA II.T-DNA I was used as template in A–C and T-DNA II in D–F. The highest dilution step in which the presence of nuclear DNA was revealed using both nuclear and numt-specific primers was step 3 (10−3, B, C) and step 4 (10−4) for mtDNA primers (A) on T-DNA I. For T-DNA II, dilution step 1 (100) was the highest dilution showing residual nuclear and numt amplification and step 5 (10−4) for mtDNA. Controls (ctrl) are no template controls in A–D and positive controls in E–F. M: DNA ladder (100 bp).
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pone-0037142-g001: Test for the presence of mitochondrial and nuclear DNA in serial dilution series' T-DNA I & T-DNA II.T-DNA I was used as template in A–C and T-DNA II in D–F. The highest dilution step in which the presence of nuclear DNA was revealed using both nuclear and numt-specific primers was step 3 (10−3, B, C) and step 4 (10−4) for mtDNA primers (A) on T-DNA I. For T-DNA II, dilution step 1 (100) was the highest dilution showing residual nuclear and numt amplification and step 5 (10−4) for mtDNA. Controls (ctrl) are no template controls in A–D and positive controls in E–F. M: DNA ladder (100 bp).

Mentions: To exclude contamination of our dataset with numts during de novo sequencing of the Black Field Cricket mtDNA, we prepared a ten-fold dilution series of the DNA extract to determine the DNA concentration revealing good amplification using mtDNA primers and only residual amplification at most of nuclear DNA (Figure 1). Consequently, dilution step 3 (10−3) was identified as suitable level of dilution for template DNA for subsequent RCA. We then prepared another ten-fold dilution series using the RCA product and used this as template for standard PCR using both mtDNA and nuclear primers to control for the presence of residual nuclear DNA and to confirm the selective enrichment of mtDNA (Figure 1). To test the general applicability of the selective enrichment of mtDNA, this strategy was also employed successfully on a whole genomic DNA extract from mammalian blood (Figure S5).


Selective enrichment and sequencing of whole mitochondrial genomes in the presence of nuclear encoded mitochondrial pseudogenes (numts).

Wolff JN, Shearman DC, Brooks RC, Ballard JW - PLoS ONE (2012)

Test for the presence of mitochondrial and nuclear DNA in serial dilution series' T-DNA I & T-DNA II.T-DNA I was used as template in A–C and T-DNA II in D–F. The highest dilution step in which the presence of nuclear DNA was revealed using both nuclear and numt-specific primers was step 3 (10−3, B, C) and step 4 (10−4) for mtDNA primers (A) on T-DNA I. For T-DNA II, dilution step 1 (100) was the highest dilution showing residual nuclear and numt amplification and step 5 (10−4) for mtDNA. Controls (ctrl) are no template controls in A–D and positive controls in E–F. M: DNA ladder (100 bp).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351421&req=5

pone-0037142-g001: Test for the presence of mitochondrial and nuclear DNA in serial dilution series' T-DNA I & T-DNA II.T-DNA I was used as template in A–C and T-DNA II in D–F. The highest dilution step in which the presence of nuclear DNA was revealed using both nuclear and numt-specific primers was step 3 (10−3, B, C) and step 4 (10−4) for mtDNA primers (A) on T-DNA I. For T-DNA II, dilution step 1 (100) was the highest dilution showing residual nuclear and numt amplification and step 5 (10−4) for mtDNA. Controls (ctrl) are no template controls in A–D and positive controls in E–F. M: DNA ladder (100 bp).
Mentions: To exclude contamination of our dataset with numts during de novo sequencing of the Black Field Cricket mtDNA, we prepared a ten-fold dilution series of the DNA extract to determine the DNA concentration revealing good amplification using mtDNA primers and only residual amplification at most of nuclear DNA (Figure 1). Consequently, dilution step 3 (10−3) was identified as suitable level of dilution for template DNA for subsequent RCA. We then prepared another ten-fold dilution series using the RCA product and used this as template for standard PCR using both mtDNA and nuclear primers to control for the presence of residual nuclear DNA and to confirm the selective enrichment of mtDNA (Figure 1). To test the general applicability of the selective enrichment of mtDNA, this strategy was also employed successfully on a whole genomic DNA extract from mammalian blood (Figure S5).

Bottom Line: We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts.Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence.This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia. jonciwolff@yahoo.com

ABSTRACT
Numts are an integral component of many eukaryote genomes offering a snapshot of the evolutionary process that led from the incorporation of an α-proteobacterium into a larger eukaryotic cell some 1.8 billion years ago. Although numt sequence can be harnessed as molecular marker, these sequences often remain unidentified and are mistaken for genuine mtDNA leading to erroneous interpretation of mtDNA data sets. It is therefore indispensable that during the process of amplifying and sequencing mitochondrial genes, preventive measures are taken to ensure the exclusion of numts to guarantee the recovery of genuine mtDNA. This applies to mtDNA analyses in general but especially to studies where mtDNAs are sequenced de novo as the launch pad for subsequent mtDNA-based research. By using a combination of dilution series and nested rolling circle amplification (RCA), we present a novel strategy to selectively amplify mtDNA and exclude the amplification of numt sequence. We have successfully applied this strategy to de novo sequence the mtDNA of the Black Field Cricket Teleogryllus commodus, a species known to contain numts. Aligning our assembled sequence to the reference genome of Teleogryllus emma (GenBank EU557269.1) led to the identification of a numt sequence in the reference sequence. This unexpected result further highlights the need of a reliable and accessible strategy to eliminate this source of error.

Show MeSH