Collaboration and Data Sources

We received 41 samples of RNA extracted from mosquito pools that tested positive for West Nile virus from Denise Bolton, Abigail Mathewson, Carolyn Fredett, Amy Kutschke and Rebecca Lovell at the New Hampshire Division of Public Health Services, Department of Health and Human Services. The samples were spread across four years: 2 from 2013, 2 from 2015, 9 from 2017 and the remaining from 2018. We were able to sequence the complete coding region of the viral genome  from all 41 samples. In this update, we highlight the sequencing results and a few conclusions drawn from a maximum likelihood based phylogenetic analysis.

Data Generation

The sequencing data was generated using PrimalSeq (Grubaugh et al. Genome Biology 2019). Our full protocol is available online here. Sequenced reads were aligned using bwa and processed using iVar.

Raw Data

Consensus sequences and BAM files along with associated metadata are available on Google Cloud. Alignment statistics are shown in Table 1.

 

Table 1. Alignment statistics
Name Percent coverage at a minimum depth of 10 Average depth per nucleotide
W830_L1.sorted.bam 99.5194 2897.05
W826_L1_L2.sorted.bam 89.7452 1824.97
W828_L1_L2.sorted.bam 95.9833 5204.15
W827_L1_L2.sorted.bam 96.2463 5749.02
W825_L1.sorted.bam 99.2746 5615.95
W829_L1_L2.sorted.bam 93.9704 5031.84
W824_L1.sorted.bam 99.2746 6060.01
W0960_L1.sorted.bam 99.3834 2703.57
W0953_L1.sorted.bam 99.3834 3807.76
W0959_L1.sorted.bam 99.3834 4837.44
W0952_L1.sorted.bam 99.3834 5426.26
W0962_L1.sorted.bam 98.9301 4903.42
W0964_L1.sorted.bam 99.3834 5783.27
W0955_L1.sorted.bam 98.6853 5536.62
W0954_L1.sorted.bam 99.3472 5104
W0951_L1.sorted.bam 99.3834 5530.52
W0961_L1.sorted.bam 98.6853 5681.15
W0956_L1.sorted.bam 89.6727 3738.55
W0950_L1.sorted.bam 98.8757 5124.33
W0957_L1.sorted.bam 99.3834 5695.19
W0958_L1.sorted.bam 99.3834 5555.3
W0965_L1.sorted.bam 98.6853 6179.75
W0963_L1.sorted.bam 99.3834 5948.02
W0966_L1.sorted.bam 99.0389 5921.09
W0967_L1.sorted.bam 98.6944 4447.62
W0968_L1.sorted.bam 99.0389 5918.12
W0969_L1.sorted.bam 99.057 5746.22
W0972_L1.sorted.bam 97.8783 5214.78
W0975_L1.sorted.bam 99.3834 5505.91
W0970_L1.sorted.bam 99.3834 5336.29
W0971_L1.sorted.bam 99.3834 5630.81
W0976_L1.sorted.bam 98.6853 5860.87
W0977_L1.sorted.bam 99.3834 5125.68
W0973_L1.sorted.bam 98.6853 5268.41
W0974_L1.sorted.bam 97.8783 4972.23
W0979_L1.sorted.bam 96.1193 4546.19
W0981_L1.sorted.bam 97.9509 5041.67
W0978_L1.sorted.bam 99.3834 5676.35
W0980_L1.sorted.bam 96.2463 5162.74
W0983_L1.sorted.bam 99.3834 5261.99
W0982_L1.sorted.bam 99.3834 5296.17
W0984_L1.sorted.bam 99.3925 5837.21
W0985_L1.sorted.bam 98.2319 5078.8
W0986_L1.sorted.bam 99.3925 5394.15
W0990_L1.sorted.bam 99.3834 4552.8
W0988_L1.sorted.bam 99.3834 5870.9
W0987_L1.sorted.bam 99.3834 5547.53
W0989_L1.sorted.bam 98.6944 5572.72

Preliminary Analysis

We constructed a maximum likelihood(ML) phylogeny using 1753 genomes of West Nile virus from USA including 41 genomes from New Hampshire (highlighted in forest green). Tree, root to tip regression plot are shown in Fig 1.

Figure 1: A. Maximum likelihood tree constructed using RAxML (Bootstrap repeated 100 times) with 1753 genomes of West Nile virus from USA including 41 genomes from New Hampshire (highlighted in forest green). B. Root to tip regression plot. C. Legend

Most of the sequences cluster with sequences from New York state but they form separate clades indicating that there have been multiple introductions into New Hampshire over the years. Given the current sampling, it’s hard to pin point if those introductions might have come from New York. W0971 from 2018-08-16 clusters along with sequences from San Diego county in California sampled in 2015 with a bootstrap support value is 77. W0953 clusters with sequences from Texas sampled in 2012 but has a very low bootstrap support value of 20. Further analysis required to see where this sequence might end up in the tree. Sequences W0967, W0968 and W0969 were identical. All three were sampled from Hillsborough county either on 2018-08-13 or 2018-08-14.

Disclaimer

Please note that this data is released as work in progress by the WestNile 4K Project and should be considered preliminary. If you intend to include any of these data in publications, please let us know – otherwise please feel free to download and use without restrictions. We have shared this data with the hope that people will download and use it, as well as scrutinize it so we can improve our methods and analyses. Please contact us if you have any questions or comments.