Major version notes

Runs prior to February 2020 had a variety of formats and sample submission requirements. Suggested that individual runs be consulted on utbox for information about each run as things changed between each run.

Overview

It is highly suggested that you join the #sequencing channel on slack to be made aware of upcoming runs.

NGS plasmid sequencing is performed on an iSeq 100 owned by the Moran lab. Illumina dual indexed libraries are prepared using 2S Turbo kits from Swift Biosciences. We currently use up to 384 6-bp indexes taken from UT's Genome Sequencing and Analysis Facility (GSAF) per run. This could be expanded significantly if throughput increases. DNA samples are due to Dan ~2-3 days before the run starts (typically Monday), and data uploaded to box ~1-2 days after the run (typically Friday).

People often ask when the next run will be and the answer is always: "When a high priority project needs to be done, or when there are enough samples on the sign up sheet". In the event that there is a high priority project, there may not be a lot of notice given for you to prepare your own samples so it is recommended that you add samples to the list as soon as you have isolated DNA and that you drop off well labeled tubes as soon as possible to secure your spot on the next available run.

Digital information and formatting

In order to sequence a plasmid, you must enter the following information onto the signup sheet on box:
  1. Tube label
    This should match the labels that you put on the PCR strip tube(s). This should be very short but unique. Currently most people go for their initials, a label for the strip, and the numbers 1-8 for each tube on the strip.
    warning if this is not unique, you risk having your samples confused with someone else's and make more work for other people.
  2. Your name
    This aids in identifying your samples in the freezer and determines how analyzed data is organized after the run. In some cases it may be better to list a group or project rather than your own name.
  3. Sample name
    This name will end up on the individual fastq files, and thus should be specific and ideally link back to the strain database.
  4. Plasmid reference (required for breseq analysis)
    If listed (and uploaded to the UTbox folder) you will receive a report detailing how the actual plasmid sequence is different than what you expected. Currently accepted formats are fasta, genbank, and gff3 with genbank or gff3 formats being preferred.
  5. Genome reference (suggested for breseq analysis)
    Plasmid preps are not perfect and thus are typically contaminated with some amount of genomic DNA. By listing the strain you grew the plasmid in, you decrease the false positive mutations reported by increasing read mapping accuracy. Currently accepted formats are fasta, genbank, and gff3 with genbank or gff3 formats being preferred.

Data analysis options

  1. Variant detection
    if you provide a plasmid reference file, breseq analysis will be performed and comparison tables generated for each reference file listed to quickly compare multiple preps.
  2. assembly
    do novo assembly of reads using plasmidSPAdes.

DNA isolation

Sample Requirements and Drop-off

-- Main.DanielDeatherage - 13 Feb 2020

 Barrick Lab  >  ProtocolList  >  ProceduresPlasmidSequencing

Contributors to this topic edittopic DanielDeatherage
Topic revision: r1 - 13 Feb 2020 - 22:27:55 - Main.DanielDeatherage
 
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