Journal: Nature Microbiology

Article Title: Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance

doi: 10.1038/s41564-019-0637-9

Figure Lengend Snippet: a , An algorithm for the design of viral spiked primers (SP). Sets of viral reference genomes ( n = 60–3,571 for each virus) were aligned using MAFFT multiple sequence alignment software , followed by the partitioning of each genome into 300–500-nt overlapping segments. Forward and reverse 13-nt primers were selected and filtered according to specific criteria (rounded rectangular box). Unique primer sequences are individually coloured in red, blue, orange and green. Using this algorithm, primers were designed for 15 RNA viruses. SP panels for ArboV ( n = 4), HFV ( n = 6) and CombV ( n = 13, excluding HCV and JCV SP) were also constructed. b , The metagenomic sequencing workflow. MSSPE primers (red) were added (spiked) to a reaction mix containing random primers (blue) during the reverse transcription step of cDNA synthesis, without adding to the overall turnaround time for the subsequent transposase-based library amplification with adapter primers (brown) and sequencing analysis protocols. The MSSPE workflow is compatible with subsequent enrichment using tiling multiplex PCR and/or capture probes (dashed lines). Metagenomic sequence data were analysed for pathogen identification using SURPI (ref. ; also see Methods). MARV, Marburg virus; RVFV, Rift Valley fever virus; HEV, hepatitis E virus; and Tm, melting temperature.

Article Snippet: Tiling multiplex PCR for ZIKV was negative when testing a contrived ZIKV sample containing the 1947 prototype Uganda strain MR766, probably due to sequence divergence from the American ZIKV strains from the 2015–2018 outbreak used in the initial multiplex PCR primer design .

Techniques: Virus, Sequencing, Software, Construct, Reverse Transcription, cDNA Synthesis, Library Amplification, Multiplex Assay

Journal: Nature Microbiology

Article Title: Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance

doi: 10.1038/s41564-019-0637-9

Figure Lengend Snippet: a , Genome coverage of the ZIKV MRC766 (Uganda) strain (mapped to accession no. LC002520 ) at 1,000 cp ml −1 with no enrichment (top) or MSSPE enrichment using ZIKV SP (second from top), an ArboV SP panel (third from top) or a CombV SP panel (bottom). With no enrichment, there were 50 reads and 45% coverage; with ZIKV SP, there were 456 reads and 97.6% coverage; with ArboV SP, 528 reads and 100% coverage; with CombV SP, there were 254 reads and 93.9% coverage. b , Genome coverage of an HIV-1 Group M, CRF01 strain (mapped to accession no. KY580709 ) at 1,000 cp ml −1 with no enrichment (left) or using HIV-1 SP (right). With no enrichment, there were 35 reads and 23.2% coverage; with HIV-1 SP, there were 289 reads and 92.8% coverage. c , Genome coverage of an HCV genotype 4 strain (mapped to accession no. KM587625 ) at 10,000 cp ml −1 with no enrichment (left) or using HCV SP (right). With no enrichment, there were 63 reads and 31.5% coverage; with HCV SP, there were 686 reads and 80% coverage. d , Genome coverage of a POWV strain identified in CSF from an infected patient with tick-borne meningoencephalitis (mapped to accession no. NC_003687 ) at <1,000 cp ml −1 with no enrichment (left) or using the ArboV SP panel (right). With no enrichment, there were 48 reads and 37.1% coverage; with ArboV SP, there were 209 reads and 88.0% coverage. e , Genome coverage of a contrived sample of LASV (Josiah strain) spiked into donor plasma matrix at a titre of 10 cp ml −1 (mapped to accession nos. AY628202 and NC_004296 ) with no enrichment (left) or using the HFV SP panel (right). With no enrichment, there were 4 reads and 3.8% coverage; with HFV SP, there were 154 reads and 67.9% coverage. f , Genome coverage of a contrived sample of CCHFV (mapped to accession nos. AY389508 , U39455 and U88410 ) spiked into donor plasma matrix at a titre of 2,500 cp ml −1 with no enrichment (left) or using the HFV SP panel (right). With no enrichment, there were 69 reads and 23.3% coverage; with HFV SP, there were 2,636 reads and 100% coverage. g , Genome coverage of a strain from a patient from Mexico with acute ZIKV infection during the 2013–2016 outbreak (ZIKV/ Homo sapiens /MEX/2016/mex30; mapped to accession no. KX879603 ) at ~2,000 cp ml −1 with no enrichment (top) or enrichment using MSSPE with ZIKV SP (second from top), tiling multiplex PCR (third from top), capture probes (fourth from top, using random primers alone) or MSSPE with ZIKV SP followed by capture probes (bottom). With no enrichment, there were 33 reads and 26.5% coverage; with ZIKV SP, there were 260 reads and 87.5% coverage; with tiling multiplex PCR, there were 158,243 reads and 88.2% coverage (75.0% ≥10× coverage); with capture probes, there were 49,927 reads and 49.1% coverage (29.6% ≥10× coverage); and with ZIKV SP plus capture probes, there were 275,105 reads and 99.8% coverage (95.6% ≥10× coverage). The red bars below the coverage plots show nucleotide regions with coverage of ≥10×, at a threshold to minimize the inclusion of cross-contaminating reads . For each graph in a – g , the number of reads is normalized to the total number of viral reads obtained with no enrichment. bp, base pairs; L, large segment; M, medium segment, S, small segment.

Article Snippet: Tiling multiplex PCR for ZIKV was negative when testing a contrived ZIKV sample containing the 1947 prototype Uganda strain MR766, probably due to sequence divergence from the American ZIKV strains from the 2015–2018 outbreak used in the initial multiplex PCR primer design .

Techniques: Infection, Clinical Proteomics, Multiplex Assay