Table 1.
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] |
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.