Difference: ProceduresPlasmidSequencing (1 vs. 3)

Major version notes

Overview

Digital information and formatting

1. 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.

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.
   • This also includes comparison tables comparing all samples that used the same reference sequence.

1. 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

DNA isolation

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.
2. 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

Major version notes

Overview

Digital information and formatting

1. 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.
2. 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.
3. 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.

DNA isolation

Sample Requirements and Drop-off

-- Main.DanielDeatherage - 13 Feb 2020

Major version notes

Overview

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

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.
   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