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 and their coordination.

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 dual indexed primers taken from UT's Genome Sequencing and Analysis Facility (GSAF) that allow up to 384 6-bp indexes 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 fully analyzed data uploaded to box ~1-2 days after the run (typically Friday). Prepping 96 samples takes ~6 hours, the run itself takes ~18 hours, and analysis can take as little as 1-2 hours meaning that if project requires it can go from DNA to data in a little over 25 hours but such turn arounds are either even more cost ineffective due to wasted space on the run, or requires significant coordination.

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. A "next run" folder will be created when submission deadline passes, and that folder will be updated to a run date when that run is scheduled. Please be sure to be putting information into the correct folder, as samples mistakenly entered into past runs have been overlooked in previous runs:
  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 such as this: Picture example to be added
    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. Raw_Reads
    raw fastq files, and tacc location returned to you for you to analyze on your own.
  2. Trimmed Reads
    fastq files as above, but with adapter sequences already removed.
  3. 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.
    • This also includes comparison tables comparing all samples that used the same reference sequence.
  4. de novo Assembly
    de novo assembly of reads using plasmidSPAdes. Does not require a reference file to be listed, will return 1 or more contigs.
    1. Future improvements
      • PLanotate - annotate the newly assembled plasmid
      • Ref comparison - blast assembled contig(s) against plasmid return a score to see how similar they are
      • Contig collapse - currently multiple different combinations of trimmed reads are used to assemble independently, many time these are equivalent sequences and thus should only be reported once.
  5. All
    breseq and de novo assembly
  6. All + Raw
    breseq and de novo assembly and Raw Reads.
    • please only select this if you can envision a situation where you will do additional advanced analysis on your own.

Data retrieval

Data will be uploaded to the same UT box directory as the sign up sheet for each run, under an analysis folder. Zip files will be returned 1 per researcher name listed on the signup sheet. Raw files will also be found on TACC in the barricklab corral-rep location (remember if you access files in this location, do not rename, or move them, only copy them to scratch or download them to your own computer.

DNA isolation

Plasmid should generally be isolated from overnight culture keeping in mind that only ~15 ng of DNA is needed. Plasmid DNA should be eluted in water or elution buffer and quantified using broad range qbit.

Sample Requirements and Drop-off

  • At least 6 Ál of 2.5 ng/Ál. 4Ál will be used for prep, additional volume will not be returned to you.
  • Samples should go into PCR strip tubes, and the strip cut down to the number of samples you have as the first thing i will do is assemble them like a jigsaw puzzle on a 96 well block and cutting out random numbers of tubes for each strip is time consuming on my end.
  • If you are generating a set of samples over time for the same run it would be preferred that you wait till you have at least 8 to submit any rather than submitting 5 on one day and then another 3 a week later.
  • Samples should be added to the racks in the B lab freezer here: PICTURE

FAQ

  1. When is the next run starting?
    • "When a high priority project needs to be done, or when there are enough samples on the sign up sheet" is always the answer. 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.
  2. What if my sample is less concentrated than 2.5 ng/Ál?
    • This is somewhat up to you. 2.5 ng/Ál is a pretty low concentration of DNA even for low copy plasmids so there is concern that if you are lower than this the plasmid prep itself may be bad or you eluted in way too high a volume. Concentrations between 0.25 and 1.25 ng/Ál may be able to be sequenced, but this has not been systematically investigated. Concentrating the plasmid DNA using the speed vac in the A lab alcove is a good choice (particularly if you eluted in water). Things to remember: 1. there is often sample loss (ie reducing 100 Ál to 10 Ál will raise the concentration by somewhat less than 10x); 2. if you eluted in a buffer originally you are increasing the salt concentration at the same time and this can lower sequencing success; 3. if you remove 100% of the liquid and then re-suspend the DNA in water is very difficult and can result in significantly lower concentrations than expected.

-- Main.DanielDeatherage - 13 Feb 2020

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Contributors to this topic edittopic DanielDeatherage
Topic revision: r2 - 09 Apr 2020 - 19:46:24 - Main.DanielDeatherage
 
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