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A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

Waugh C, Cromer D, Grimm A, Chopra A, Mallal S, Davenport M, Mak J - Virol. J. (2015)

Bottom Line: Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase.We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts.Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Deakin University and CSIRO(AAHL), Geelong, VIC, Australia. caryll.waugh@csiro.au.

ABSTRACT

Background: Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles.

Method: Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses.

Results: We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts.

Conclusions: Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

No MeSH data available.


Related in: MedlinePlus

Measurement of recombination and mutation rates. (Top) Alignment of GAG1 region of HIV-1 WT sequence (pDRNL(AD8)) (top line) and corresponding marker sequence (bottom line) showing the 15 silent mutations introduced into adjacent codons. This created 14 intervals spaced approximately 50 nucleotides apart (range 18 to 156 nucleotides). Numbering is based on the pDRNL(AD8) WT sequence. Dots indicate identical sequence and silent mutations are indicated with orange highlight. (Bottom) Schematic representation of the marker system depicting silent markers (red) and positions of the overlapping amplicons (brown bars) that enable detection of recombination events (Adapted from [20].
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Fig6: Measurement of recombination and mutation rates. (Top) Alignment of GAG1 region of HIV-1 WT sequence (pDRNL(AD8)) (top line) and corresponding marker sequence (bottom line) showing the 15 silent mutations introduced into adjacent codons. This created 14 intervals spaced approximately 50 nucleotides apart (range 18 to 156 nucleotides). Numbering is based on the pDRNL(AD8) WT sequence. Dots indicate identical sequence and silent mutations are indicated with orange highlight. (Bottom) Schematic representation of the marker system depicting silent markers (red) and positions of the overlapping amplicons (brown bars) that enable detection of recombination events (Adapted from [20].

Mentions: The wild-type (WT) HIV-1 plasmid used was pDRNL(AD8) [19] that encodes an R5-tropic strain of HIV-1. A modified, ‘marker’ version of this plasmid (MK) was created by introducing 15 pairs of silent genetic marker points by nucleotide substitution into gag, as previously described [1,20,21] and depicted in Figure 6. This created a total of 13 intervals spaced, on average about 50 nucleotides apart where recombination can be measured. Importantly, the marker points did not change the protein profile of the virus, the replication kinetics compared to wild-type (WT) virus or known RNA sequence elements [1,20,21]. It is important to note that this system does not introduce ‘foreign’ genes into the HIV-1 genome, such as fluorescent markers, antibiotic resistance genes or surface protein markers that can drastically change the RNA structure. Furthermore, the series of silent mutations enables detection of recombination events over a significant length of the genome (Figure 6) and unlike other systems, also permits the identification of multiple template switches. A full description of our system and its attendant advantages in accurately measuring recombination has been published [7,20].Figure 6


A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

Waugh C, Cromer D, Grimm A, Chopra A, Mallal S, Davenport M, Mak J - Virol. J. (2015)

Measurement of recombination and mutation rates. (Top) Alignment of GAG1 region of HIV-1 WT sequence (pDRNL(AD8)) (top line) and corresponding marker sequence (bottom line) showing the 15 silent mutations introduced into adjacent codons. This created 14 intervals spaced approximately 50 nucleotides apart (range 18 to 156 nucleotides). Numbering is based on the pDRNL(AD8) WT sequence. Dots indicate identical sequence and silent mutations are indicated with orange highlight. (Bottom) Schematic representation of the marker system depicting silent markers (red) and positions of the overlapping amplicons (brown bars) that enable detection of recombination events (Adapted from [20].
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403950&req=5

Fig6: Measurement of recombination and mutation rates. (Top) Alignment of GAG1 region of HIV-1 WT sequence (pDRNL(AD8)) (top line) and corresponding marker sequence (bottom line) showing the 15 silent mutations introduced into adjacent codons. This created 14 intervals spaced approximately 50 nucleotides apart (range 18 to 156 nucleotides). Numbering is based on the pDRNL(AD8) WT sequence. Dots indicate identical sequence and silent mutations are indicated with orange highlight. (Bottom) Schematic representation of the marker system depicting silent markers (red) and positions of the overlapping amplicons (brown bars) that enable detection of recombination events (Adapted from [20].
Mentions: The wild-type (WT) HIV-1 plasmid used was pDRNL(AD8) [19] that encodes an R5-tropic strain of HIV-1. A modified, ‘marker’ version of this plasmid (MK) was created by introducing 15 pairs of silent genetic marker points by nucleotide substitution into gag, as previously described [1,20,21] and depicted in Figure 6. This created a total of 13 intervals spaced, on average about 50 nucleotides apart where recombination can be measured. Importantly, the marker points did not change the protein profile of the virus, the replication kinetics compared to wild-type (WT) virus or known RNA sequence elements [1,20,21]. It is important to note that this system does not introduce ‘foreign’ genes into the HIV-1 genome, such as fluorescent markers, antibiotic resistance genes or surface protein markers that can drastically change the RNA structure. Furthermore, the series of silent mutations enables detection of recombination events over a significant length of the genome (Figure 6) and unlike other systems, also permits the identification of multiple template switches. A full description of our system and its attendant advantages in accurately measuring recombination has been published [7,20].Figure 6

Bottom Line: Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase.We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts.Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Deakin University and CSIRO(AAHL), Geelong, VIC, Australia. caryll.waugh@csiro.au.

ABSTRACT

Background: Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles.

Method: Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses.

Results: We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts.

Conclusions: Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

No MeSH data available.


Related in: MedlinePlus