2G Analyser | Second-generation sequencing

Work area

clean work:
- powder-free gloves;
- lens cleaning tissue;
- 1L bottle with mQ water;
- washing bottles with clean
- 100ml glass beaker for rinsing tubes;
boxes for storage of caps:
- reagent bottles;
- PE-reagent bottles;
- washing bottles;
instruments:
- plastic tweezers;
- empty flowcell for NaOH washing;
- 1ml and 200µl tips;
- 250µl positive/displacement automatic pipette with special tips for oil loading;
- tube holders: 50ml, 15ml, 2ml;
solutions:
- "clean ethanol" in 2ml aliquotes;
- working aliquote of mineral oil;
writing:
- marker;
- pen;

Priming lines

positions 1-7 on GAII: [X · 250 · 2500 · 100];
PE module positions 9-21:
- 12 times: 9,10,11,12,13
- 15 times: 14,15,16,17,18,19,20
- 19 times: 21

Maintenance

before each run: Pre-run wash;
after each run: Post-run wash and empty the waste bottle;
every 4 weeks or when GAII is not used for more than 3 days: NaOH wash;
general practice:
- protect GAII and the PE module from dust:
  wash the outer and inner surfaces with wet paper towel every time GAII is used; use only lens cleaning tissues for washing the imaging compartment; vacuum-clean the sequencing room every three months;
- whenever you change tubes on GAII, do it in the order: 2,1,3,4,5,7,6 (cleavage mix — the last);

Pre-run wash: SINGLE RUN / PE runPE RUN / single run

Use FC from the previous run. Wash takes ~15min for single-run~40min for PE-run.

prepare bottles with mQ water
- 4x 250ml with ~40ml of mQ for positions 4,5,2,7;
- 3x 50ml with ~10ml of mQ for positions 1,6,3;
- 13x 15ml with ~12ml of mQ for positions 9-21 on the PE module;
substitute old water bottles/tubes with fresh ones;

NB! check the tubing connections: if they are not tight air would be pumped during the run

place bundled waste tubings in the 50ml waste tube;
open "GA2_PreWash_v4" recipe ⇒ Start;
open "GA2-PEM_PreWash_v4" recipe ⇒ Start;

positions 1-7: [X · 250 · 2500 · 125]

positions 9-21: [X · 60 · 2000 · 125]

record the waste volume: should be ~7ml for single-run~20ml for PE-run;

Post-run wash: SINGLE RUN / PE runPE RUN / single run

Wash takes ~20min for single-run~60min for PE-run.

NB! Wash the tubing from a washing bottle (with air pumping) when replace reagent bottles/tubes for water tubes.

substitute reagent tubes/bottles in the positions 1-7 with water tubes/bottles from the pre-run wash in order: 1,3,4,5,7,6:
- unscrew the reagent tube;
- wash the tubing with a washing bottle during air pumping [X · 60 · 2000 · 30] ⇒ Enter;
- screw up the water tube;
- change water in the positions 9-21 on the PE module (reagents were substituted for water tubes after PE read 2);
place bundled waste tubings in the 50ml waste tube;
open "GA2_PostWash_v4" recipe ⇒ Start;
open "GA2-PEM_PostWash_v4" recipe ⇒ Start;

positions 1-7: [X · 250 · 2500 · 500]

positions 9-21: [X · 60 · 2000 · 125]

record the waste volume: should be ~28ml for single-run~41ml for PE-run;

NaOH wash

a special "cleaning FC" for NaOH wash;
~800ml of mQ water;
1N NaOH (Merck, already filtered), ~40ml in a glass beaker;
100ml glass beaker for rinsing ends of tubes;
new water tubes (w/o signing):
- 3x 50ml tubes with 40ml H₂O: positions 1,6,3;
- 4x 250ml bottles with 100ml H₂O: positions 4,5,2,7;
- 13x 15ml tubes with 10ml H₂O: positions 9-21

NB! Take care to avoid contact of NaOH with caps and tubing of GAII and PE module.

empty waste container, rinse it with water;
change water for fresh one in positions 1-7 on the GAII and 9-21 on the PE module;
install the "cleaning FC":
- open "Manual Control/Setup" ⇒ "Load Flowcell";
- "Instrument" ⇒ "Unlock door";
- pump air gap: [to: flowcell · solution: 28 · volume: 0 · asp. rate: 250 · disp. rate: 2500] ⇒ Enter;
- lift the manifolds;
- remove the old FC:
  blot excess of oil with lens cleaning tissue;
  remove the rest of the oil with lens cleaning tissue wetted with ethanol;
  clean FC with water-wetted lens cleaning tissue;
  put FC into storage buffer;
- remove the prism:
  blot excess of oil with lens cleaning tissue;
  remove the rest of the oil under stream of ethanol;
  clean the prism with water-wetted lens cleaning tissue;
- clean the imaging compartment:
  
  Peltier element;
  FC table;
  beam dump;
  manifolds (water only);
  remove dust from reachable surfaces with lens cleaning tissue wetted with ethanol;
- load the prism & "cleaning FC";
- lower manifolds;
reagent delivery check:
- place the bundled waste tubings in a 1.7ml tube;
- pump water to check bubbles: [to: flowcell · solution: 5 · volume: 100 · asp. rate: 60 · disp. rate: 2500] ⇒ Enter;
- if bubbles are coming from the inlet manifold — reinstall the FC;
- check the waste volume, should be 720-880µl;
open the "GA2-PEM_MaintenanceWash_v4" recipe ⇒ Start;
click OK to start the prewash:
positions 1-7: [X · 250 · 2500 · 125];
positions 9-21: [X · 60 · 2000 · 125]
when finish click Cancel;
manual NaOH wash
- discard the old tube;
- put the tubing (~4cm) into NaOH and wash it: [X · 250 · 2500 · 125] ⇒ Enter;
- rinse the tubing using washing bottle during air gap pumping: [X · 250 · 2500 · 50] ⇒ Enter;
- attach new water tube;
resume the "GA2-PEM_MaintenanceWash_v4" recipe from the "PostNaOHWash" step.

GA II

Fig. 1. Reagents. 1 — waste; 2 — syringe pump; 3 — bottles at room temperature (4: high salt buffer, 5: incorporation buffer, 2: H₂O, 7: cleavage buffer); 4 — tubes at 4°C (1: incorporation mix, 6: cleavage mix, 3: scan mix); 5 — valve.

Fig. 2. Flowcell table 1 — prism; 2 — input tube; 3 — output tubes; 4 — objective; 5 — laser; 6 — manifold lever; 7 — laserbeam catcher; 8 — flowcell.

Flowcell

Fig. 3. Flowcell (old model).

Fig. 4. Scheme of image frames 200 images are taken from the central part of each chanell. All distances in µm.

Old-type of FlowCell is describes here

flowcell size: 66 x 17 x 1,4 mm (Fig. 3);
number of channels: 8;
size of each channel: length: 61mm; image zone: ~36mm; width: 1mm (bridle: 0,5mm); depth: ~0,1mm (for microscope ~75µm); volume: ~7µl;
images: 2 rows per channel, 100 per row (Fig. 3a);
image size: ~340 x 340µm; 1002 x 1004px (Fig. 3a)
FC channels should not be dried out: contact WET holes with lens cleaning tissue VERY ACCURATELY!!! (especially if FC is oriented vertically). If some channell will dry out, put a drop of High Salt Buffer on the hole with 200µl pipet. Channell will be filled by capillary force;
Contact clean flowcell and prism only with clean(!) gloves or with lens cleaning tissue. Do not rub surfaces, clean it by blotting or one-movement cleaning;

Flowcell storage

5x SSC for short-term storage
20x SSC for long-term storage

Notes

Immersion oil: Cargille Labs, Cartille Immersion liquid, Cat: #custom, Qty: 1 fl.oz., n=1,4730±0,0005 R.I.Units, wavelength 5893 Å Temp 25°C, Code 50350, Lot: 072482.

GAII works under the negative pressure. There should be no leakage except for one during connection/disconnection of tubing. The problem associated with leakage — dirt on flowcell or FC-manifold would change the focal plane of the glass. This would result in uneven tile-lightening and bad focusing.

The problem associated with bubbles — clusters are invisible through the bubble. Sign of problems — bubbles in the waste tubes. To localize the problem open the instrument door, pump some "neutral" buffer through the FC, and try to localize in what part of the system bubbles appear. If bubbles appear in the flowcell input, slightly relax manifold and accurately push the flowcell in the correct position (upper-right). If it does not help — take out the flowcell, clean it again, install it again. If the prism was disturbed — reclean and reinstall the prism.