Difference: ProceduresPlasmidSequencing (1 vs. 2)

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
   if this is not unique, you risk having your samples confused with someone else's and make more work for other people.

1. 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.

1. 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.
2. All
   breseq and de novo assembly
3. 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

• 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.