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add data from mumps-seq-library-4, update protocols to include inform…

…ation about Maganpure and full nextera reactions, fix epiweek/strain names in old genomes, and update former library sequences to reflect a 20x required coverage cutoff for calling a consensus base rather than a 100x coverage cutoff
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lmoncla committed Dec 20, 2019
1 parent dc7a968 commit c364807f60e7a461ba71d41914cf9df4b041b003

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@@ -21,10 +21,10 @@ We used a [genome](https://www.ncbi.nlm.nih.gov/nuccore/MF965301) from the mumps
The mapping (bam) file was manually inspected in [Geneious](https://www.geneious.com/).

### Consensus sequence calling
Consensus sequences were called in Geneious, with nucleotide sites with <100x coverage called as Ns. Consensus genomes were exported in fasta format.
Consensus sequences were called in Geneious, with nucleotide sites with <20x coverage called as Ns. Consensus genomes were exported in fasta format.

### Remapping
To avoid issues with mapping to improper reference sequences, we then remapped each sample's fastq files to its own consensus sequence. These bam files were again manually inspected in Geneious, and a final consensus sequence was called. Those consensus genomes for which we acquired at least 80% full-genome coverage are available [here](https://github.com/blab/mumps-seq/tree/master/data/consensus-genomes) as fasta files.
To avoid issues with mapping to improper reference sequences, we then remapped each sample's fastq files to its own consensus sequence. These bam files were again manually inspected in Geneious, and a final consensus sequence was called. Those consensus genomes for which we acquired at least 50% full-genome coverage are available [here](https://github.com/blab/mumps-seq/tree/master/data/consensus-genomes) as fasta files.



@@ -1,6 +1,6 @@
# Library preparation

Library preparation was performed with the Nextera XT DNA Library Prep kit (Illumina, catalogue # FC-131-1096), following the manufacturer's recommended protocol, but with reagent volumes halved for each step.
Library preparation was performed with the Nextera XT DNA Library Prep kit (Illumina, catalogue # FC-131-1096), following the manufacturer's recommended protocol. For sequencing runs 1-3, we halved the reagent volumes for each step. For sequencing run 4, we had quite a few samples with high Ct values and a few samples that were old (from 2006), so we used full reaction volumes. The protocol below is for the half reaction volumes. To perform the full reaction (as specified in the Illumina recommended protocol), double the volumes and concentrations for each step. You can also follow this [protocol](https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera-xt/nextera-xt-library-prep-reference-guide-15031942-02.pdf) from the prep kit manual.

### Pooling amplicons from pool 1 and pool 2
1. For each sample, pool 1 and pool 2 amplicons were combined in equimolar concentrations to a total of 0.5 ng in 2.5 µl in a PCR strip tube. This generally required diluting the cleaned PCR product, re-quantifying, and using the diluted product for pooling. All reactions were brought up to 2.5 µl with water.
@@ -1,5 +1,7 @@
# vRNA extraction

For sequencing runs 1-3, we extracted viral RNA from buccal swabs manually, using the protocol described below. For sequencing run 4, we had access to a Roche Magnapure RNA extraction robot, which we used. Both methods produced good quality RNA. For the manual extraction, we have provided the protocol below.

This protocol follows the published protocol for vRNA extraction with the QiAmp viral RNA mini kit (Qiagen, catalogue # 52906). Most mumps samples are collected as buccal (cheek) swabs, which are likely to contain host cells and proteins. To remove host cells and maximize the amount of material that we could extract, we added 2 additional spin steps to the beginning of the standard protocol. The first spin pellets out and removes host cells. The second spin concentrates virions at the bottom of the tube. Excess supernatant is removed and the virions are resuspended in the ~140-200 ul necessary for the start of the protocol, concentrating the sample. All parts of this protocol should be carried out in a clean, pre-PCR hood that has been wiped down with bleach and ethanol before use. Until particle lysis is complete (step 5), there are infectious virions in the sample. All pipette tips and tubes should be discarded and soaked in 10% bleach for 1 hour to sterilize.

### Concentrate vRNA and remove host cells

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