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 (40Â bp region of interest) | Longitudinal emergence of drug resistance during treatment failure | [10] |
HIV | RT, PCR amplificatin of 4 fragments (3.5Â 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] |
76Â 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] |