Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

Abstract

Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/ sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS. IMPORTANCEAlthough next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set. Studies of viral population diversity are increasingly using nextgeneration sequencing (NGS) technologies to extend the depth of population sampling. Key aspects of understanding within-host viral population diversity are knowing the true depth of template/genome sampling and documenting the accuracy of the sequencing method to validate the detection of rare variants. Current approaches using NGS in viral population studies usually require a preceding PCR amplification step. Thus, PCR errors, including nucleotide misincorporation ...

Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions

“…cDNA synthesis was done by using a primer that included a 9-nucleotide stretch of randomized bases, an indexing strategy that we have termed Primer ID (32,33); the Primer ID cDNA primer was used just for cDNA synthesis and then removed prior to the PCR step. This allowed each RNA template to be linked to a unique sequence tag that could be identified in a subsequent sequence analysis.…”

“…To avoid the inclusion of sequencing errors in the Primer ID sequence tag (offspring Primer IDs) that would create artifactual viral genomes, we calculated a cutoff for the number of raw reads needed for each Primer ID to form a template consensus sequence (TCS) based on a simulation to estimate the frequency of offspring Primer IDs with errors. We then used Primer IDs with a number of raw reads above this cutoff to create a TCS for each starting RNA template (33). Additionally, TCSs were filtered for large deletions and frameshifts.…”

“…We used the QIAamp viral RNA minikit (Qiagen, Valencia, CA) to extract viral RNA from the plasma samples and a previously described approach to construct the Primer ID MiSeq library after cDNA synthesis using the viral RNA as the template and PCR (33). The Primer ID cDNA primer was composed of the complement of the target region in the RNA template for cDNA synthesis (HXB2 [GenBank accession number K03455] numbering for the gene-specific region spanning positions 7209 to 7238, at the 3= boundary of the V3 coding region of the env gene), an 8-base random sequence that served as the Primer ID and one random nucleotide as a sequencing buffer, and a 5= tail of the primer sequence that served as the PCR primer.…”

“…Artifactual heterogeneity can be introduced due to the preceding PCR step, which introduces misincorporation and recombination (29,30) as well as high rates of sequencing errors associated with NGS, while artifactual homogeneity can be introduced by PCR resampling of the same starting templates (31) and using reference sequences to construct consensus sequences for variable regions. We have overcome these limitations by using the Primer ID sequencing approach to define template sampling depth and reduce the error rate to around 1 in 10,000 nucleotides (32,33).…”

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

“…We used the previously described protocol (25,26) to generate the amplicon for the viral protease coding domain as Primer ID sequencing libraries to sequence the antisense strand of HIV-1 protease gene from the incubated ssDNA described above, with the following modifications. We used Platinum Taq HiFi DNA polymerase (Life Technology) for first-strand synthesis of the ssDNA template using the Primer ID primer instead of using reverse transcriptase.…”