High-throughput, short-read (Illumina) sequencing using a MiSeq i100 (Plus)
I’ve been using the original Illumina Miseq system for my small sequencing runs (amplicon libraries to assess on-target editing, small screens, validating WGS & (sc)RNA-seq libraries before deep sequencing) over the past 5 years but recently our genomics core acquired the new Illumina Miseq i100 Plus system and it’s amazing. I wanted to share details on how to load/run it since it’s so much easier, faster, and cheaper and I figure it’ll be quite popular for the next few years. This also allows me a space to walk through Illumina’s chemistry.
Why do I like this instrument so much? It’s a huge improvement over the original MiSeq in all the categories that matter (sequencing yield, kit cost, loading time, runtime). For example, it used to cost $1100 for a 5M read, 300-cycle kit which would take 1-2hrs to load (including the time it took for the reagents to thaw) and 24hr to sequence. With the new Miseq i100, for $550, I can purchase a 5M read, 300-cycle kit which takes <30 min to load (no thawing required) and 8hr to sequence. Essentially, everything improved 2-4x. That’s real progress.
Chemistry:
There is already a great online resource summarizing Illumina adaptors and sequencing chemistry (https://teichlab.github.io/scg_lib_structs/methods_html/Illumina.html) but I’ll summarize briefly here focusing on the Miseq i100 approach (which mirrors the NovaSeq 6000).
The core idea is we need to add universal ends to our DNA of interest so that’s it’s “sequenceable” on Illumina’s instruments. In other words, we need to adapt our DNA. Along the way, we also add unique sequences (“indexes”) to the DNA to mark (“barcode”) separate samples so that we can pool and sequence a bunch of DNA together (“multiplex”) then assign individual reads back to their original source later. There’s a lot of jargon when it comes to sequencing but it’s all fairly sensible once you understand the underlying process.
Loading:
Decide on which kit you want to run (see table on right or Illumina's website for more details)
Available kits generate 5M or 25M quality clusters (reads that pass filter) with 100-, 300-, and 600-cycle options. 50-100M read kits coming soon for the i100 Plus which is a good size to validate cloned pools and/or small CRISPR screens.
Kits composed of two separate components (dry reagent package and wet reagent box)
Retrieve package of dry reagents and set aside
Contains single, large cartridge containing flow cell and sample loading wells.
Stored at room temp. so no need to thaw or keep on ice
Note the letter ("A" or "B") on the package indicating which wet reagent box you need.
Retrieve box of wet reagents
Contains 2 small vials of buffers on top for sample prep and a larger, sealed container of sequencing reagents/buffers
Stored at room temp. so no need to thaw or keep on ice
Make sure letter ("A" or "B") on top/front matches dry reagent package
Open wet reagent box to retrieve vials of RSB (resuspension) and KLD (denaturation) buffers
Prepare (at least) 25 uL soln of pooled libraries at 1 nM (1000 pM) total in resuspension buffer (RSB). Once made, briefly vortex and spin.
An example from my test run (“HTS_RAM_080"):
3.33 uL of diluted HTS_RAM_80 sample (3 nM)
5 uL of Phi-X (1 nM... loosely)
16.7 uL of resuspension buffer (RSB)
This means I now have a solution where my library is at a 0.4 nM concentration and phi-X is at 0.2 nM concentration for a total of 0.6 nM (600 pM)
Illumna recommends a 1 nM (1000 pM) library here which then gets diluted 0.1x in KLD buffer for a final loading concentration = 100 pM but this needs adjusting based on the library type, especially for small amplicon libraries
Dilute sample 0.1x by adding 225 of KLD buffer to 25 uL of "1 nM" sample. Briefly vortex and spin.
Now "100 pM" and all ready to load (actually 60 pM for my test run)
Next, open dry reagent package containing flow cell (no need to wash).
Pierce foil for "library" well (leftmost well) and load all 250 uL of prepared sample (denatured library at “100 pM”)
Other wells are for custom primers, if using. Apparently need to dilute in hybridization buffer which you now need to buy from Illumina separately (doesnt come w/ the i100 kits)
Bring everything (loaded flow cell and wet reagent cassette) to Miseq i100 instrument, sign in, enter run parameters, and load
Note: Should DESELECT the option to read indexes first so that Miseq i100 operates "normally" (as old MiSeq used to) and reads R1 first before i7 then i5 then R2
Load dry reagent cartridge first, follow graphic to ensure correct orientation
Load wet reagent cartridge second, follow graphic to ensure correct orientation
Instrument takes ~10-20 min to perform system checks then proceeds automatically into run
Don't need to return to instrument to start after system checks
Once running, Miseq i100 provides estimated time of completion (4-14h depending on kit and #-cycles)
Will take 2-3hr from start of run for instrument to provide estimate on cluster density
Took 6.5h total from start (run started) to finish (data uploaded) for 25M, 100-cycle kit
My test run generated 35M reads total with 5M from phi-X (14%) from a loading concentration of 60 pM
Checking with our core director, it appears 30-50 pM is the sweet spot for amplicon libraries +/- phi-X
NOTE(1): KLD buffer is quite nasty/toxic so EHS recommends double gloving and discarding all consumables that touch it in a special, designated waste bottle for later pickup.
NOTE(2): MiSeq i100 has a waste container on the right side that need emptying after every few runs, check before loading/running.
Once signing into instrument w/ username & password on right side of instrument go to menu > settings > open side door
Empty contents of container into labeled, secondary waste container right next to instrument for EHS to pickup and discard