Opened 6 years ago

Closed 6 years ago

#762 closed task (fixed)

Pre-normalization changes due to NeoPrep

Reported by: Nicklas Nordborg Owned by: Nicklas Nordborg
Priority: major Milestone: Reggie v3.4
Component: net.sf.basedb.reggie Keywords:
Cc:

Description (last modified by Nicklas Nordborg)

The neoprep require only 100ng of input rna in 25-45µl. The current pre-normalization is hard-wired for the manual protocol which is optimized for 1.1µg in 50µl.

The pre-normalization need to be modified so that it also works with the NeoPrep. The required RNA quantity and volume should be selectable, with at least the following presets:

  • The old (dUTP) method (1.1µg in 50µl)
  • TruSeq manual method (0.5µg in 50µl)
  • NeoPrep method (0.1µg in 45µl)

Due to the much lower quantity of RNA for NeoPrep the pre-normalization will need to support mixing a larger volume (to avoid volumes less than 1µl). This also means that the mRNA wizard need to be aware of this, since it currently assumes that everything is used from the pre-normalized aliquot.

Another issue with the mRNA wizard is that if there are existing pre-normalized RNA for the NeoPrep and the mRNA wizard is used with the intention of creating a manual TruSeq plate, the NeoPrep samples will be auto-selected and then flagged with NotEnoughRemainingQuantity (correction, they will not be flagged, but included on the mRNA plate and the lab protocol will display a very large volume in the RNA column). This makes it very difficult the various protocols unless all queues are processed so they are empty before filling with RNA for a different protocol. Clearly the current auto-select function in mRNA wizard need to be much smarter about what to auto-select. The wizard also need to know about the various library preparation protocols. A separate ticket has been created for solving this issue (#773).

Change History (12)

comment:1 Changed 6 years ago by Nicklas Nordborg

Milestone: Reggie v3.xReggie v3.4

comment:2 Changed 6 years ago by Nicklas Nordborg

Description: modified (diff)
Summary: Pre-normalization changes due to neoprepPre-normalization changes due to NeoPrep

comment:3 Changed 6 years ago by Nicklas Nordborg

Status: newassigned

comment:4 Changed 6 years ago by johan

TruSeq? manual method is (0.5µg in 50µl)

comment:5 Changed 6 years ago by Nicklas Nordborg

Description: modified (diff)

comment:6 Changed 6 years ago by Nicklas Nordborg

Description: modified (diff)

comment:7 Changed 6 years ago by Nicklas Nordborg

Description: modified (diff)

comment:8 Changed 6 years ago by Nicklas Nordborg

Description: modified (diff)

comment:9 Changed 6 years ago by Nicklas Nordborg

One idea is to introduce a new protocol type (Normalization) and two annotation types (RNATargetAmount and RNATargetVolume). The server admin should create protocols for the three options above with proper annotations. The "Project default" can be used to set the default selected protocol. The selected protocol is then saved as the creation protocol for the normalized aliquot.

Another advantage with the method is that the protocol can then be used as a filter criteria for the auto-select function in the mRNA wizard so that it doesn't select RNA that was pre-normalized for a different protocol.

comment:10 Changed 6 years ago by Nicklas Nordborg

(In [3269]) References #762: Pre-normalization changes due to NeoPrep?

Added new Normalization protocol type. At least one protocol need to be created to be able to use this wizard. All normalization protocols should be annotated with RNATargetAmount (µg) and RNATargetVolume (µl) so that the normalization wizard can calculate amount of RNA and water to use when mixing.

The volume of RNA can't be less than 1µl. If the RNA concentration is high a larger mix may have to be created to reach the desired normalized concentration.

comment:11 Changed 6 years ago by Nicklas Nordborg

(In [3273]) References #762: Pre-normalization changes due to NeoPrep?

The mRNA registration wizard now register a separate event for "large mixes" of pre-normalized RNA to make sure that the remaining quantity reaches 0 after it has been used.

comment:12 Changed 6 years ago by Nicklas Nordborg

Resolution: fixed
Status: assignedclosed
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