Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations – application to HIV-1 quasispecies
Creators: name: Westfall, Dylan
name: James, Mullins
Publisher: name: Zenodo
Year: 2023
Subjects: PacBio sequencing
DNA barcodes
Julia
R Code

description: Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciences single molecule real-time platform was used to sequence PCR amplicons derived from cDNA templates tagged with universal molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR and the use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Handling of the large datasets produced from SMRT-UMI sequencing was facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline), that automatically filters and parses reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination or early cycle PCR errors, resulting in highly accurate sequence datasets. The optimized SMRT-UMI sequencing method presented here represents a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus (HIV) quasispecies.
description: Sequence analysis pipelines, R code, and supplemental info for these data are found at:



https://github.com/MullinsLab/chunked_demux.git

https://github.com/MullinsLab/sUMI_dUMI_comparison.git

https://doi.org/10.5281/zenodo.7672191

https://doi.org/10.5281/zenodo.7672189

Funding provided by: United States Public Health ServiceCrossref Funder Registry ID: https://ror.org/05xf94514Award Number: UM1 AI06818
Funding provided by: University of Washington/Fred Hutch Centers for AIDS Research*Crossref Funder Registry ID: Award Number: P30 AI027757
description: This serves as an overview of the analysis performed on PacBio sequence data that is summarized in Analysis Flowchart.pdf and was used as primary data for the paper by Westfall et al. "Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations – application to HIV-1 quasispecies"

Five different PacBio sequencing datasets were used for this analysis: M027, M2199, M1567, M004, and M005

For the datasets which were indexed (M027, M2199), CCS reads from PacBio sequencing files and the chunked_demux_config files were used as input for the chunked_demux pipeline. Each config file lists the different Index primers added during PCR to each sample. The pipeline produces one fastq file for each Index primer combination in the config. For example, in dataset M027 there were 3–4 samples using each Index combination. The fastq files from each demultiplexed read set were moved to the sUMI_dUMI_comparison pipeline fastq folder for further demultiplexing by sample and consensus generation with that pipeline. More information about the chunked_demux pipeline can be found in the README.md file on GitHub.

The demultiplexed read collections from the chunked_demux pipeline or CCS read files from datasets which were not indexed (M1567, M004, M005) were each used as input for the sUMI_dUMI_comparison pipeline along with each dataset's config file. Each config file contains the primer sequences for each sample (including the sample ID block in the cDNA primer) and further demultiplexes the reads to prepare data tables summarizing all of the UMI sequences and counts for each family (tagged.tar.gz) as well as consensus sequences from each sUMI and rank 1 dUMI family (consensus.tar.gz). More information about the sUMI_dUMI_comparison pipeline can be found in the paper and the README.md file on GitHub.

The consensus.tar.gz and tagged.tar.gz files were moved from sUMI_dUMI_comparison pipeline directory on the server to the Pipeline_Outputs folder in this analysis directory for each dataset and appended with the dataset name (e.g. consensus_M027.tar.gz). Also in this analysis directory is a Sample_Info_Table.csv containing information about how each of the samples was prepared, such as purification methods and number of PCRs. There are also three other folders: Sequence_Analysis, Indentifying_Recombinant_Reads, and Figures. Each has an `.Rmd` file with the same name inside which is used to collect, summarize, and analyze the data. All of these collections of code were written and executed in RStudio to track notes and summarize results.

`Sequence_Analysis.Rmd` has instructions to decompress all of the consensus.tar.gz files, combine them, and create two fasta files, one with all sUMI and one with all dUMI sequences. Using these as input, two data tables were created, that summarize all sequences and read counts for each sample that pass various criteria. These are used to help create Table 2 and as input for `Indentifying_Recombinant_Reads.Rmd` and `Figures.Rmd`. Next, 2 fasta files containing all of the rank 1 dUMI sequences and the matching sUMI sequences were created. These were used as input for the python script compare_seqs.py which identifies any matched sequences that are different between sUMI and dUMI read collections. This information was also used to help create Table 2. Finally, to populate the table with the number of sequences and bases in each sequence subset of interest, different sequence collections were saved and viewed in the Geneious program.

To investigate the cause of sequences where the sUMI and dUMI sequences do not match, tagged.tar.gz was decompressed and for each family with discordant sUMI and dUMI sequences the reads from the UMI1_keeping directory were aligned using geneious. Reads from dUMI families failing the 0.7 filter were also aligned in Genious. The uncompressed tagged folder was then removed to save space. These read collections contain all of the reads in a UMI1 family and still include the UMI2 sequence. By examining the alignment and specifically the UMI2 sequences, the site of the discordance and its case were identified for each family as described in the paper. These alignments were saved as "Sequence Alignments.geneious". The counts of how many families were the result of PCR recombination were used in the body of the paper.

Using `Identifying_Recombinant_Reads.Rmd`, the dUMI_ranked.csv file from each sample was extracted from all of the tagged.tar.gz files, combined and used as input to create a single dataset containing all UMI information from all samples. This file dUMI_df.csv was used as input for Figures.Rmd.

`Figures.Rmd` used dUMI_df.csv, sequence_counts.csv, and read_counts.csv as input to create draft figures and then individual datasets for eachFigure. These were copied into Prism software to create the final figures for the paper.