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Table 1 Representative examples of deep sequencing applied to viral populations

From: Deep sequencing of evolving pathogen populations: applications, errors, and bioinformatic solutions

Pathogen Design Technology Ref seq Filter Align SNV Hap Application Reference
HIV RT-PCR, nested PCR of pol fragment Roche-454 GS-FLX amplicon sequencing Sanger sequenced pol gene In-house software: removes reads with ambiguous bases, < 80% similarity to reference, or outside region of interest GS amplicon software (Roche, Penzberg, Germany), Needleman-Wunsch In house scripts, manual inspection: remove gaps, remove reads with frameshift indels or stop codons, remove variants only contained in reads in one direction, positional variant cut-off values based on control sequences Individual reads (40 bp region of interest) Longitudinal emergence of drug resistance during treatment failure [10]
HIV RT, PCR amplificatin of 4 fragments (3.5 kb each). Full genome analysis Roche-454 GS-FLX Titanium De novo assembled reference using AssembleViral454 v1.0 NS Mosaik RC454 / V-Phaser V-Phaser (one read length only) Longitudinal emergence of CD8+ T cell escape variants, viral adaptation [11]
HCV RT, PCR amplification of HVR-1, nested PCR using sequencing adapters Roche-454 GS-FLX Titanium amplicon sequencing 358 HCV HVR-1 representative sequences from Los Alamos National Laboratory HCV Flow clustering as implemented in QIIME, only reads covering entire region of interest MAFFT (multiple sequence alignment) NA Individual reads Identification of a transmission event [12]
HCV Whole-genome library prep direct from RNA isolated from human serum, using mRNA-seq sample prep kit (Illumina, San Diego, CA) Illumina GA IIx 76 bp single end reads 970 reference HCV sequences registered at the Hepatitis Virus Database server Primer stripping using CLC Genomics Workbench (4.6), remove reads aligning to human genome, removal of duplicate reads BWA 0.5.9-r16 Samtools (0.1.16) NA PCR-free whole genome HCV sequencing from human serum; variant comparison between treatment naïve and treatment experienced patients [13]
HCV RT-PCR using genotype specific primers, nested PCR of full genome, followed by random shearing and library preparation Roche-454 GS-FLX Titanium Sanger-sequenced consensus In house software (discard reads with Phred quality scores below 20 or length < 55nt) Mosaik ShoRAH, manual cleaning ShoRAH (up to 1600 bp reconstructions) Within-host evolution/genetic bottleneck [14]
HRV Duplicate whole-genome RT-PCR of overlapping primer pairs, nebulisation of pooled fragments and library prep Illumina GA IIx Sanger-sequenced consensus Illumina software: RTA SCS.2.6 and CASAVA 1.6 MAQ v0.7.1 In house scripts; cut-off based on statistical analyses of base frequencies along reference. Comparison between replicates. NA Within-host evolution during immunosuppression [15]
76 bp single end reads
Dengue RT, PCR amplification of four different fragments, random shearing and adapter ligation Roche-454 GS-FLX Titanium De novo assembled using AV454 with manual finishing NS Mosaik RC454/ V-Phaser. Manual removal of variants in primer binding sites or only in ends of reads NA Intra-host viral diversity [16]
Poliovirus RT-PCR and nested PCR of target amplicon, followed by random shearing and library preparation Roche-454 FLX Titanium and Illumina GA IIx 76 bp single end reads Known amplicon sequences Proprietary Roche/Illumina software. In house software (discard reads with Phred quality scores below 20). NS Custom made scripts – disregard variants with strand bias, as well as insertions and deletions adjacent to homopolymers for Roche-454 data. NA Detection of emerging resistant variants in a vaccine stock [17]
  1. Details of the experimental design and analysis pipeline for various applications of deep sequencing to different viruses are given. ‘Design’ describes the types of samples used and any sample processing up to library preparation. ‘Technology’ indicates the type of sequencing employed. ‘Filter’ details any pre-alignment read processing steps. ‘Ref. Seq.’ describes what kinds of reference sequences were used for read alignment, while ‘Align’ gives the actual alignment software used. ‘SNV’ and ‘Hap.’ indicate software used for SNV detection and haplotype reconstruction respectively. ‘Application’ describes the biological motivation for the study. ‘NS’ indicates the method was not specified in the cited publication, while ‘NA’ means not attempted.