onetaq one step rt pcr kit  (New England Biolabs)


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    New England Biolabs onetaq one step rt pcr kit
    Optimization of <t>RT-PCR</t> <t>kit</t> and parameters for the generation of SARS-CoV-2 cDNA for genome sequencing. (A) RT-PCR of 18 SARS-CoV-2 RNA samples was performed using the <t>two-step</t> Lunascript + Q5 protocol or the Takara 1-step kit. For 9 of 18 samples, the 1-step kit performed better at generating a visible band than the 2-step protocol. (B) 384 SARS-CoV-2 positive RNA samples were sequenced using both the 2-step and Takara 1-step protocols. In addition to generating more reconstructed genomes, the Takara 1-step protocol generated fewer uncalled bases (B) , better average genome coverage (C) and longer consensus genomes (D) than the 2-step protocol. RT-PCR was also performed on 37 samples using either 1μL or 5μL of sample as input in the reaction. The 5μL reactions were found to have ( E ) fewer uncalled bases, ( F ) greater average coverage, and ( G ) longer average genome length than achieved in the 1μL cases.
    Onetaq One Step Rt Pcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/onetaq one step rt pcr kit/product/New England Biolabs
    Average 94 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    onetaq one step rt pcr kit - by Bioz Stars, 2022-08
    94/100 stars

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    1) Product Images from "Development and scaling of a sequencing pipeline for genomic surveillance of SARS-CoV-2 in New York City"

    Article Title: Development and scaling of a sequencing pipeline for genomic surveillance of SARS-CoV-2 in New York City

    Journal: medRxiv

    doi: 10.1101/2022.05.25.22273991

    Optimization of RT-PCR kit and parameters for the generation of SARS-CoV-2 cDNA for genome sequencing. (A) RT-PCR of 18 SARS-CoV-2 RNA samples was performed using the two-step Lunascript + Q5 protocol or the Takara 1-step kit. For 9 of 18 samples, the 1-step kit performed better at generating a visible band than the 2-step protocol. (B) 384 SARS-CoV-2 positive RNA samples were sequenced using both the 2-step and Takara 1-step protocols. In addition to generating more reconstructed genomes, the Takara 1-step protocol generated fewer uncalled bases (B) , better average genome coverage (C) and longer consensus genomes (D) than the 2-step protocol. RT-PCR was also performed on 37 samples using either 1μL or 5μL of sample as input in the reaction. The 5μL reactions were found to have ( E ) fewer uncalled bases, ( F ) greater average coverage, and ( G ) longer average genome length than achieved in the 1μL cases.
    Figure Legend Snippet: Optimization of RT-PCR kit and parameters for the generation of SARS-CoV-2 cDNA for genome sequencing. (A) RT-PCR of 18 SARS-CoV-2 RNA samples was performed using the two-step Lunascript + Q5 protocol or the Takara 1-step kit. For 9 of 18 samples, the 1-step kit performed better at generating a visible band than the 2-step protocol. (B) 384 SARS-CoV-2 positive RNA samples were sequenced using both the 2-step and Takara 1-step protocols. In addition to generating more reconstructed genomes, the Takara 1-step protocol generated fewer uncalled bases (B) , better average genome coverage (C) and longer consensus genomes (D) than the 2-step protocol. RT-PCR was also performed on 37 samples using either 1μL or 5μL of sample as input in the reaction. The 5μL reactions were found to have ( E ) fewer uncalled bases, ( F ) greater average coverage, and ( G ) longer average genome length than achieved in the 1μL cases.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Generated

    Increasing effective concentration of SARS-CoV-2 cDNA during tagmentation and in the final sequenced libraries improves sequencing outcomes. (A) RT-PCR was performed on a set of 288 SARS-CoV-2 positive samples which were then tagmented under standard conditions or with 8% PEG 8000 and 10-fold reduced transposase concentration. Samples tagmented with PEG in general achieved greater reads per sample, and 16 genomes reconstructed only in the PEG+ condition (green), compared to only 6 in the PEG-case (orange). (B) Reconstructed samples from this experiment were plotted by Ct and colored by whether genome reconstruction succeeded in both treatments (light blue), only in the PEG-condition(red), or only in the PEG+ condition (dark blue). Samples which reconstruct only in the PEG+ case are highly biased toward high-Ct samples, suggesting that these samples may preferentially reconstruct due to molecular crowding induced by PEG. (C) Samples from the PEG- and PEG+ conditions are displayed in violin plots of the number of uncalled bases with individual points represented. There is no statistically significant difference in the overall number of uncalled bases per sample. (D) 384 samples above Ct 26.5 were sequenced either with or without hybrid capture of barcoded libraries. Samples which were reconstructed in both conditions are labeled TRUE, samples which were reconstructed in neither are labeled FALSE, and samples reconstructed only when hybrid capture was applied are labeled SAVED. Saved samples are biased toward higher Ct. (E) Reconstructed genome length is plotted for both samples to which hybrid capture was and was not applied. Genome lengths are longer when samples are treated with hybrid capture. (F) Uncalled bases are plotted for both samples to which hybrid capture was and was not applied. Samples have fewer uncalled bases on average when hybrid capture is applied.
    Figure Legend Snippet: Increasing effective concentration of SARS-CoV-2 cDNA during tagmentation and in the final sequenced libraries improves sequencing outcomes. (A) RT-PCR was performed on a set of 288 SARS-CoV-2 positive samples which were then tagmented under standard conditions or with 8% PEG 8000 and 10-fold reduced transposase concentration. Samples tagmented with PEG in general achieved greater reads per sample, and 16 genomes reconstructed only in the PEG+ condition (green), compared to only 6 in the PEG-case (orange). (B) Reconstructed samples from this experiment were plotted by Ct and colored by whether genome reconstruction succeeded in both treatments (light blue), only in the PEG-condition(red), or only in the PEG+ condition (dark blue). Samples which reconstruct only in the PEG+ case are highly biased toward high-Ct samples, suggesting that these samples may preferentially reconstruct due to molecular crowding induced by PEG. (C) Samples from the PEG- and PEG+ conditions are displayed in violin plots of the number of uncalled bases with individual points represented. There is no statistically significant difference in the overall number of uncalled bases per sample. (D) 384 samples above Ct 26.5 were sequenced either with or without hybrid capture of barcoded libraries. Samples which were reconstructed in both conditions are labeled TRUE, samples which were reconstructed in neither are labeled FALSE, and samples reconstructed only when hybrid capture was applied are labeled SAVED. Saved samples are biased toward higher Ct. (E) Reconstructed genome length is plotted for both samples to which hybrid capture was and was not applied. Genome lengths are longer when samples are treated with hybrid capture. (F) Uncalled bases are plotted for both samples to which hybrid capture was and was not applied. Samples have fewer uncalled bases on average when hybrid capture is applied.

    Techniques Used: Concentration Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Labeling

    PRL integrated pipeline for COVID-19 testing and sequencing. The integrated pipeline begins with RNA extraction and RT-qPCR-based testing of incoming patient samples at the InnoLabs facility. After categorizing samples as either PCR-positive or negative, residual RNA from samples which are positive for SARS-CoV-2 RNA are automatically identified and reformatted, or “hitpicked”, into 384-well plates for processing in our sequencing pipeline. The positive samples are then converted to DNA using multiplexed RT-PCR, and tagmentation-based library prep and barcoding is performed on the resulting cDNA. Samples are then pooled separately into low Ct and high Ct bins, and the samples in the high Ct bin go through an extra step of hybrid capture before being sequenced on an appropriate Illumina platform. A parallel workflow to directly accept specimens from external facilities (the STATseq line) was also established. Specimens diagnosed as positive by the external platform are extracted in the R D facility and fed directly into the sequencing pipeline. See Materials and Methods for more detail on all steps of the pipeline.
    Figure Legend Snippet: PRL integrated pipeline for COVID-19 testing and sequencing. The integrated pipeline begins with RNA extraction and RT-qPCR-based testing of incoming patient samples at the InnoLabs facility. After categorizing samples as either PCR-positive or negative, residual RNA from samples which are positive for SARS-CoV-2 RNA are automatically identified and reformatted, or “hitpicked”, into 384-well plates for processing in our sequencing pipeline. The positive samples are then converted to DNA using multiplexed RT-PCR, and tagmentation-based library prep and barcoding is performed on the resulting cDNA. Samples are then pooled separately into low Ct and high Ct bins, and the samples in the high Ct bin go through an extra step of hybrid capture before being sequenced on an appropriate Illumina platform. A parallel workflow to directly accept specimens from external facilities (the STATseq line) was also established. Specimens diagnosed as positive by the external platform are extracted in the R D facility and fed directly into the sequencing pipeline. See Materials and Methods for more detail on all steps of the pipeline.

    Techniques Used: Sequencing, RNA Extraction, Quantitative RT-PCR, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    2) Product Images from "A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing"

    Article Title: A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing

    Journal: bioRxiv

    doi: 10.1101/2020.04.07.029199

    qSanger detects SARS-COV-2 RNA when amplified directly from viral particles in transport medium. ( A ) A total of 32 no-template controls, 32 negative control samples (Seracare) and 32 positive samples (Seracare) were assayed. All results were concordant with Seracare and NTC. Three samples were no-calls (undetermined) due to low signal-to-noise ratio in the sequencing results. ( B ) Positive Seracare samples were added to RT-PCR master mix either directly from the VTM or after purification with RNA extraction kit at 125 GCE. The ratio of reference and spike-in intensities were measured by custom data analysis. The mean qSanger ratio was 0.745 (± 0.043 s.e.m., n=8) for direct addition, and 0.97 (± 0.041 s.e.m., n=8) for purified. The coefficient of variation (CV) of positive seracare samples were measured for both Luna and OneTaq polymerase mixes. The CV for Luna direct VTM was 16.4% (n=8), and for Luna purified was 12.1% (n=8). This is consistent with the theoretical counting noise associated with quantifying ~100 molecules.
    Figure Legend Snippet: qSanger detects SARS-COV-2 RNA when amplified directly from viral particles in transport medium. ( A ) A total of 32 no-template controls, 32 negative control samples (Seracare) and 32 positive samples (Seracare) were assayed. All results were concordant with Seracare and NTC. Three samples were no-calls (undetermined) due to low signal-to-noise ratio in the sequencing results. ( B ) Positive Seracare samples were added to RT-PCR master mix either directly from the VTM or after purification with RNA extraction kit at 125 GCE. The ratio of reference and spike-in intensities were measured by custom data analysis. The mean qSanger ratio was 0.745 (± 0.043 s.e.m., n=8) for direct addition, and 0.97 (± 0.041 s.e.m., n=8) for purified. The coefficient of variation (CV) of positive seracare samples were measured for both Luna and OneTaq polymerase mixes. The CV for Luna direct VTM was 16.4% (n=8), and for Luna purified was 12.1% (n=8). This is consistent with the theoretical counting noise associated with quantifying ~100 molecules.

    Techniques Used: Amplification, Negative Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, Purification, RNA Extraction

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  • 94
    New England Biolabs onetaq one step rt pcr kit
    Optimization of <t>RT-PCR</t> <t>kit</t> and parameters for the generation of SARS-CoV-2 cDNA for genome sequencing. (A) RT-PCR of 18 SARS-CoV-2 RNA samples was performed using the <t>two-step</t> Lunascript + Q5 protocol or the Takara 1-step kit. For 9 of 18 samples, the 1-step kit performed better at generating a visible band than the 2-step protocol. (B) 384 SARS-CoV-2 positive RNA samples were sequenced using both the 2-step and Takara 1-step protocols. In addition to generating more reconstructed genomes, the Takara 1-step protocol generated fewer uncalled bases (B) , better average genome coverage (C) and longer consensus genomes (D) than the 2-step protocol. RT-PCR was also performed on 37 samples using either 1μL or 5μL of sample as input in the reaction. The 5μL reactions were found to have ( E ) fewer uncalled bases, ( F ) greater average coverage, and ( G ) longer average genome length than achieved in the 1μL cases.
    Onetaq One Step Rt Pcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/onetaq one step rt pcr kit/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    onetaq one step rt pcr kit - by Bioz Stars, 2022-08
    94/100 stars
      Buy from Supplier

    95
    New England Biolabs one taq one step rt pcr
    Optimization of <t>RT-PCR</t> <t>kit</t> and parameters for the generation of SARS-CoV-2 cDNA for genome sequencing. (A) RT-PCR of 18 SARS-CoV-2 RNA samples was performed using the <t>two-step</t> Lunascript + Q5 protocol or the Takara 1-step kit. For 9 of 18 samples, the 1-step kit performed better at generating a visible band than the 2-step protocol. (B) 384 SARS-CoV-2 positive RNA samples were sequenced using both the 2-step and Takara 1-step protocols. In addition to generating more reconstructed genomes, the Takara 1-step protocol generated fewer uncalled bases (B) , better average genome coverage (C) and longer consensus genomes (D) than the 2-step protocol. RT-PCR was also performed on 37 samples using either 1μL or 5μL of sample as input in the reaction. The 5μL reactions were found to have ( E ) fewer uncalled bases, ( F ) greater average coverage, and ( G ) longer average genome length than achieved in the 1μL cases.
    One Taq One Step Rt Pcr, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/one taq one step rt pcr/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    one taq one step rt pcr - by Bioz Stars, 2022-08
    95/100 stars
      Buy from Supplier

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    Optimization of RT-PCR kit and parameters for the generation of SARS-CoV-2 cDNA for genome sequencing. (A) RT-PCR of 18 SARS-CoV-2 RNA samples was performed using the two-step Lunascript + Q5 protocol or the Takara 1-step kit. For 9 of 18 samples, the 1-step kit performed better at generating a visible band than the 2-step protocol. (B) 384 SARS-CoV-2 positive RNA samples were sequenced using both the 2-step and Takara 1-step protocols. In addition to generating more reconstructed genomes, the Takara 1-step protocol generated fewer uncalled bases (B) , better average genome coverage (C) and longer consensus genomes (D) than the 2-step protocol. RT-PCR was also performed on 37 samples using either 1μL or 5μL of sample as input in the reaction. The 5μL reactions were found to have ( E ) fewer uncalled bases, ( F ) greater average coverage, and ( G ) longer average genome length than achieved in the 1μL cases.

    Journal: medRxiv

    Article Title: Development and scaling of a sequencing pipeline for genomic surveillance of SARS-CoV-2 in New York City

    doi: 10.1101/2022.05.25.22273991

    Figure Lengend Snippet: Optimization of RT-PCR kit and parameters for the generation of SARS-CoV-2 cDNA for genome sequencing. (A) RT-PCR of 18 SARS-CoV-2 RNA samples was performed using the two-step Lunascript + Q5 protocol or the Takara 1-step kit. For 9 of 18 samples, the 1-step kit performed better at generating a visible band than the 2-step protocol. (B) 384 SARS-CoV-2 positive RNA samples were sequenced using both the 2-step and Takara 1-step protocols. In addition to generating more reconstructed genomes, the Takara 1-step protocol generated fewer uncalled bases (B) , better average genome coverage (C) and longer consensus genomes (D) than the 2-step protocol. RT-PCR was also performed on 37 samples using either 1μL or 5μL of sample as input in the reaction. The 5μL reactions were found to have ( E ) fewer uncalled bases, ( F ) greater average coverage, and ( G ) longer average genome length than achieved in the 1μL cases.

    Article Snippet: A variety of 1-step kits were tested on extracted SARS-CoV-2 RNA samples, including the NEB OneTaq One-Step (#E5315S), Invitrogen SuperScript™ IV One-Step (#12594025), Promega GoTaq® 1-step (#A6121), and Takara One Step PrimeScript III (#RR600A) kits.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Generated

    Increasing effective concentration of SARS-CoV-2 cDNA during tagmentation and in the final sequenced libraries improves sequencing outcomes. (A) RT-PCR was performed on a set of 288 SARS-CoV-2 positive samples which were then tagmented under standard conditions or with 8% PEG 8000 and 10-fold reduced transposase concentration. Samples tagmented with PEG in general achieved greater reads per sample, and 16 genomes reconstructed only in the PEG+ condition (green), compared to only 6 in the PEG-case (orange). (B) Reconstructed samples from this experiment were plotted by Ct and colored by whether genome reconstruction succeeded in both treatments (light blue), only in the PEG-condition(red), or only in the PEG+ condition (dark blue). Samples which reconstruct only in the PEG+ case are highly biased toward high-Ct samples, suggesting that these samples may preferentially reconstruct due to molecular crowding induced by PEG. (C) Samples from the PEG- and PEG+ conditions are displayed in violin plots of the number of uncalled bases with individual points represented. There is no statistically significant difference in the overall number of uncalled bases per sample. (D) 384 samples above Ct 26.5 were sequenced either with or without hybrid capture of barcoded libraries. Samples which were reconstructed in both conditions are labeled TRUE, samples which were reconstructed in neither are labeled FALSE, and samples reconstructed only when hybrid capture was applied are labeled SAVED. Saved samples are biased toward higher Ct. (E) Reconstructed genome length is plotted for both samples to which hybrid capture was and was not applied. Genome lengths are longer when samples are treated with hybrid capture. (F) Uncalled bases are plotted for both samples to which hybrid capture was and was not applied. Samples have fewer uncalled bases on average when hybrid capture is applied.

    Journal: medRxiv

    Article Title: Development and scaling of a sequencing pipeline for genomic surveillance of SARS-CoV-2 in New York City

    doi: 10.1101/2022.05.25.22273991

    Figure Lengend Snippet: Increasing effective concentration of SARS-CoV-2 cDNA during tagmentation and in the final sequenced libraries improves sequencing outcomes. (A) RT-PCR was performed on a set of 288 SARS-CoV-2 positive samples which were then tagmented under standard conditions or with 8% PEG 8000 and 10-fold reduced transposase concentration. Samples tagmented with PEG in general achieved greater reads per sample, and 16 genomes reconstructed only in the PEG+ condition (green), compared to only 6 in the PEG-case (orange). (B) Reconstructed samples from this experiment were plotted by Ct and colored by whether genome reconstruction succeeded in both treatments (light blue), only in the PEG-condition(red), or only in the PEG+ condition (dark blue). Samples which reconstruct only in the PEG+ case are highly biased toward high-Ct samples, suggesting that these samples may preferentially reconstruct due to molecular crowding induced by PEG. (C) Samples from the PEG- and PEG+ conditions are displayed in violin plots of the number of uncalled bases with individual points represented. There is no statistically significant difference in the overall number of uncalled bases per sample. (D) 384 samples above Ct 26.5 were sequenced either with or without hybrid capture of barcoded libraries. Samples which were reconstructed in both conditions are labeled TRUE, samples which were reconstructed in neither are labeled FALSE, and samples reconstructed only when hybrid capture was applied are labeled SAVED. Saved samples are biased toward higher Ct. (E) Reconstructed genome length is plotted for both samples to which hybrid capture was and was not applied. Genome lengths are longer when samples are treated with hybrid capture. (F) Uncalled bases are plotted for both samples to which hybrid capture was and was not applied. Samples have fewer uncalled bases on average when hybrid capture is applied.

    Article Snippet: A variety of 1-step kits were tested on extracted SARS-CoV-2 RNA samples, including the NEB OneTaq One-Step (#E5315S), Invitrogen SuperScript™ IV One-Step (#12594025), Promega GoTaq® 1-step (#A6121), and Takara One Step PrimeScript III (#RR600A) kits.

    Techniques: Concentration Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Labeling

    PRL integrated pipeline for COVID-19 testing and sequencing. The integrated pipeline begins with RNA extraction and RT-qPCR-based testing of incoming patient samples at the InnoLabs facility. After categorizing samples as either PCR-positive or negative, residual RNA from samples which are positive for SARS-CoV-2 RNA are automatically identified and reformatted, or “hitpicked”, into 384-well plates for processing in our sequencing pipeline. The positive samples are then converted to DNA using multiplexed RT-PCR, and tagmentation-based library prep and barcoding is performed on the resulting cDNA. Samples are then pooled separately into low Ct and high Ct bins, and the samples in the high Ct bin go through an extra step of hybrid capture before being sequenced on an appropriate Illumina platform. A parallel workflow to directly accept specimens from external facilities (the STATseq line) was also established. Specimens diagnosed as positive by the external platform are extracted in the R D facility and fed directly into the sequencing pipeline. See Materials and Methods for more detail on all steps of the pipeline.

    Journal: medRxiv

    Article Title: Development and scaling of a sequencing pipeline for genomic surveillance of SARS-CoV-2 in New York City

    doi: 10.1101/2022.05.25.22273991

    Figure Lengend Snippet: PRL integrated pipeline for COVID-19 testing and sequencing. The integrated pipeline begins with RNA extraction and RT-qPCR-based testing of incoming patient samples at the InnoLabs facility. After categorizing samples as either PCR-positive or negative, residual RNA from samples which are positive for SARS-CoV-2 RNA are automatically identified and reformatted, or “hitpicked”, into 384-well plates for processing in our sequencing pipeline. The positive samples are then converted to DNA using multiplexed RT-PCR, and tagmentation-based library prep and barcoding is performed on the resulting cDNA. Samples are then pooled separately into low Ct and high Ct bins, and the samples in the high Ct bin go through an extra step of hybrid capture before being sequenced on an appropriate Illumina platform. A parallel workflow to directly accept specimens from external facilities (the STATseq line) was also established. Specimens diagnosed as positive by the external platform are extracted in the R D facility and fed directly into the sequencing pipeline. See Materials and Methods for more detail on all steps of the pipeline.

    Article Snippet: A variety of 1-step kits were tested on extracted SARS-CoV-2 RNA samples, including the NEB OneTaq One-Step (#E5315S), Invitrogen SuperScript™ IV One-Step (#12594025), Promega GoTaq® 1-step (#A6121), and Takara One Step PrimeScript III (#RR600A) kits.

    Techniques: Sequencing, RNA Extraction, Quantitative RT-PCR, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    qSanger detects SARS-COV-2 RNA when amplified directly from viral particles in transport medium. ( A ) A total of 32 no-template controls, 32 negative control samples (Seracare) and 32 positive samples (Seracare) were assayed. All results were concordant with Seracare and NTC. Three samples were no-calls (undetermined) due to low signal-to-noise ratio in the sequencing results. ( B ) Positive Seracare samples were added to RT-PCR master mix either directly from the VTM or after purification with RNA extraction kit at 125 GCE. The ratio of reference and spike-in intensities were measured by custom data analysis. The mean qSanger ratio was 0.745 (± 0.043 s.e.m., n=8) for direct addition, and 0.97 (± 0.041 s.e.m., n=8) for purified. The coefficient of variation (CV) of positive seracare samples were measured for both Luna and OneTaq polymerase mixes. The CV for Luna direct VTM was 16.4% (n=8), and for Luna purified was 12.1% (n=8). This is consistent with the theoretical counting noise associated with quantifying ~100 molecules.

    Journal: bioRxiv

    Article Title: A Highly Scalable and Rapidly Deployable RNA Extraction-Free COVID-19 Assay by Quantitative Sanger Sequencing

    doi: 10.1101/2020.04.07.029199

    Figure Lengend Snippet: qSanger detects SARS-COV-2 RNA when amplified directly from viral particles in transport medium. ( A ) A total of 32 no-template controls, 32 negative control samples (Seracare) and 32 positive samples (Seracare) were assayed. All results were concordant with Seracare and NTC. Three samples were no-calls (undetermined) due to low signal-to-noise ratio in the sequencing results. ( B ) Positive Seracare samples were added to RT-PCR master mix either directly from the VTM or after purification with RNA extraction kit at 125 GCE. The ratio of reference and spike-in intensities were measured by custom data analysis. The mean qSanger ratio was 0.745 (± 0.043 s.e.m., n=8) for direct addition, and 0.97 (± 0.041 s.e.m., n=8) for purified. The coefficient of variation (CV) of positive seracare samples were measured for both Luna and OneTaq polymerase mixes. The CV for Luna direct VTM was 16.4% (n=8), and for Luna purified was 12.1% (n=8). This is consistent with the theoretical counting noise associated with quantifying ~100 molecules.

    Article Snippet: qSanger Amplification Reverse transcription and amplification for was performed using OneTaq One-Step RT-PCR Kit from NEB (cat. # E5315S).

    Techniques: Amplification, Negative Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, Purification, RNA Extraction