Protocols and pipelines
Samples from the USVI
Sample metadata is specified in
Consensus genomes for USVI samples
Genome sequences that we generated from samples collected in the USVI are available in the generated-usvi-sequences directory
About the different available FASTA files
Genomes are collected into FASTAS with “good” coverage (>80% of sites have non-ambiguous base calls) in
ZIKA_USVI_complete_goodQual.fasta and with “partial” coverage (50%-80% of sites have non-ambiguous base calls) in
ZIKA_USVI_complete_partialQual.fasta. Genomes that have less than 50% coverage (over half the sites in the genomes have ambiguous basecalls) are in
While we have shared
ZIKA_USVI_complete_poorQual.fasta online, we do not use these genomes in any of our main analyses. These genomes have so little unambiguous sequence data that they will not cluster appropriately in a phylogeny.Just to note, while VI24 has slightly more than 50% unambiguous basecalls, it was sequenced on 2D chemistry after ONT pulled the kit and the bioinformatic pipelines. It is therefore not nanopolished, and we are wary of the demuxing as well as this sample was demuxed by an early version of ONT’s built-in demuxer. Given that we cannot appropriately QC this specific sequenced, we consider it to be poor quality as well.
For some samples there are multiple consensus genomes. This occurred in instances where we sequenced the sample differently, either on the MiSeq or MinION, using different MinION sequencing chemistries, or using different multiplexing strategies (either multiplexing 6 samples or 12 samples on a single run). This information is contained in the fasta header. So while a single sample might have multiple consensus genomes each fasta header is unique.
While We wanted to release all of the data we generated, for the analyses we only want to include one sequence per sample. For isolates that had multiple sequences, we selected the sequence included in our analysis according to the following criteria:
- The selected sequence must have been stringently demuxed.
- The selected sequence must have been nanopolished.
- If both of the above criteria were met for multiple sequences, then the sequence with the greatest number of informative base calls was selected to include in the analysis.
The USVI sequences used for downstream analyses (one sequence per isolate) are in
Gathering contextual Zika genomic data from other countries in Oceania and the Americas
To contextualize the USVI genomic data we need to include Zika genomes from other countries affected by the epidemic as well. Here I describe the process I used to collate those additional genomes and finalize the dataset for use in my BEAST analyses. While the phylogenetic analysis in the paper uses BEAST, I like using Nextstrain to quickly iterate through a process of finalizing a dataset, as you’ll see in my description below. These steps are implemented in make-input-fastas.ipynb.
The USVI sequences included in the analysis dataset come from
To gather available Zika genomes from other countries countries, I downloaded all Zika genomes available from Nextstrain/Fauna. Nextstrain/Fauna is Nextstrain’s internal database for canonicalizing sequence data, but it takes in genomes available on public databases such as NCBI and ViPR.
While all of these sequences were publicly available from repositories, not all of them had been included in previously published analyses. In the lab we feel that it’s important not to include unpublished data in published analyses, even if you get that data from NCBI/ViPR, unless you have permission from the sequence authors. So, I removed any sequences for which I did not receive author permission to include them, or which were not associated with a publication.
I also removed any Zika sequences that were from geographic areas that I didn’t want to include (e.g. Singapore), or were not high quality sequences of sufficient length to include in analyses. This left me with 437 sequences. These are contained within the
usvi-and-high-qual-americas-input-data.fastais my input file for my Nextstrain build that I ran to do additional dataset QC, such as looking for excessive terminal branch length, deviation from the molecular clock, etc. Using Nextstrain also allowed me to infer dates for some genomes that lacked full year/month/date information, as well as do some bioinformatics such as making the multiple sequence alignment. You can explore the Nextstrain build at: nextstrain.org/community/blab/zika-usvi.
The output file from the Nextstrain build is a JSON file (it lives within the
auspicedirectory in the top level of this repo). For the BEAST analyses in the paper I used the final dataset that is specified within
zika-usvi.json. I did need to format the data contained in the JSON so that it could be read in to Beauti to make the BEAST xml. The code that I wrote to do that parsing is also in the
The final generated dataset that is outputted from the “make-input-fastas.ipynb” notebook is a dataset of 429 aligned genomes, and it is written to
zika-beast-input-2020-02-20.fasta. This is the fasta file that I used to generate my BEAST XML files.
More detailed notes about dataset QC that I performed iteratively while exploring my Nextstrain build.
Here I outline the exclusion criteria for genomes in the analysis. Genomes that should be excluded from the phylogeographic analysis have been hardcoded in to
Nextstrain/augur in the
dropped_strains section of the
These QC steps were either carried out in
nextstrain-augur, or in my make-input-fastas.ipynb script.
1) The genome has too few informative bases.
The first constraint that I’m imposing on my alignment is that the genome must have a minimum of 5000 informative bases (ie A, C, G, or T) in order to be included in the analysis alignment.
Nextstrain/fauna grabs all publicly available sequences, so I’ve looked through these sequences and determined which genomes
2) The genome has excessive terminal branch length.
Excessive terminal branch length accrues when many mutations within a single genome are not shared with any other genomes. This is often and indication that the genome is poor quality or has many sequencing errors, which is why we remove these.
3) The genome may have contamination.
Sequencing Zika from clinical samples is challenging and prone to contamination due to generally low viral titers in PCR positive samples. Since contamination during PCR amplification is a very real risk, we remove genomes if we see possible signatures of contamination in the genome.
4) The genome deviates significantly from the molecular clock.
Pronounced deviation from the molecular clock may be indicative that the stated sampling date of the genome is incorrect, or that there may be errors in the genome. Given that temporal signal is very important to BEAST analyses, these genomes are excluded.
5) The genome is a duplicate.
Some isolates may be sequenced multiple times. We drop strains such that a single isolate is only ever represented by one genome sequence.