ProceduresPlasmidSequencing < Lab

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---++ 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 #logistics channel on slack to be made aware of upcoming runs and their coordination. 

The most common question people ask is: "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.

All libraries are generated using 2S Turbo kits from Swift Biosciences for paired end sequencing on Illumina instruments. We currently use dual indexed primers taken from UT's  [[https://wikis.utexas.edu/display/GSAF/Illumina+-+all+flavors][Genome Sequencing and Analysis Facility (GSAF)]]. We currently have 24 i7 indexes and 16 i5 indexes in combination this results in 384 6-bp different indexes, and is the maximum number of samples that are included in a single run. This could be expanded by purchasing additional indexes.

Plasmids and genomes are typically sequenced on separate runs due to the difference in number of reads per run, and size of plasmids vs genomes. As such there are always 2 different sign up sheets available, 1 for plasmids, and 1 for genomes. 

---+++Plasmid sequencing
"Plasmid Sequencing" is used here to refer to sequencing performed on DNA isolated from procedures that exclude genomic DNA. Sequencing is done on an iSeq 100 giving 2x150bp fastq files. 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. 

---+++Genome sequencing
"Genome sequencing" is used to refer to sequencing performed on DNA isolated that includes genomic DNA even if there is additional plasmid DNA present in the cells. Sequencing is done on a HighSeqX giving 2x150bp fastq files. Typically, DNA samples are due to Dan before Monday, libraries are sent out on Thursday, and results come back in ~2-4 weeks.


---++ Digital information and formatting
Both the plasmid sequencing and genome sequencing sign up sheets contain the same information.There should always be a "next PLASMID/GENOME run" folder with a signup sheet. If you have samples and don't see such a folder, contact DNA. Please be sure that you are entering your information into folders that do not have a date. Samples mistakenly entered into past runs have been overlooked in previous runs, and likely will be again.

The sign up sheet itself contains information about how to fill out each column, but more information is provided here: 
   1 *Tube label* <br> 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* <br>%ICON{"warning"}% if this is not unique, you risk having your samples confused with someone else's and make more work for other people.
   1 *Your name* <br> This 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 (such as BTK, or iGem).
   1 *Sample name* <br> This name will end up on the individual fastq files, and thus should be specific and ideally link back to the strain database.
   1 *Reference File* (required for _breseq_ analysis) <br> 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.
      1 If you have multiple reference files (such as a plasmid and a genome, list all references separated by a comma)

---++ Data analysis
   1 *Variant detection* <br> 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.
   1 __de novo__ *Assembly* <br> _de novo_ assembly of reads using unicycler. 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

---++ 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 fastq files are available if you contact Dan.

---++ 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 µl of 2.5 ng/µl. 4µl 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.
   1 *What if my sample is less concentrated than 2.5 ng/µl?*
      * This is somewhat up to you. 2.5 ng/µl 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/µl 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 µl to 10 µl 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
Barrick Lab > ProtocolList > ProceduresPlasmidSequencing
Topic revision: r3 - 2021-11-03 - 20:04:22 - Main.DanielDeatherage