Opened 6 years ago

Closed 6 years ago

#596 closed enhancement (fixed)

Import adapter peak information when registering libraries

Reported by: Nicklas Nordborg Owned by: Nicklas Nordborg
Priority: major Milestone: Reggie v2.15.2
Component: net.sf.basedb.reggie Keywords:
Cc:

Description (last modified by Nicklas Nordborg)

The library registration wizard currently import values for the library size (in 180-600BP) region and concentration measurements (from Qubit). This information is used to calculate the molarity (stored as CA_Molarity annotation).

While this is fine most of the time it can yield incorrect results (too low molarity) if the adapter peak (which is in the 120-140BP region) is high compared to the library peak.

When calculating the final molarity the size used should be a weighted average between the average adapter size and the average library size. The lab procedure has now been updated to include more information in the Caliper well table file:

  • Region[120-140] Size [BP]: The average size of the adaptors
  • Region[120-140] Molarity (nmol/l): The molarity of the adaptors (must be scaled by a factor of 10!)
  • Region[180-600] Size [BP]: The average size of the library fragments (this is the same column as before)
  • Region[180-600] Molarity (nmol/l): The molarity of the library (must be scaled by a factor of 10!)

In principle the Qubit concentration values are not needed, but since we believe that they have higher accuracy, we use the molarity values from Caliper only to calculate a weighted size and fraction of adapters:

S(w) = S(a)*M(a) + S(l)*M(l) / (M(a)+M(l))
F(a) = M(a) / (M(a)+M(l))

Then, we use the weighted size and the Qubit concentration to calculate the final molarity as before.

We need some new annotation types:

  • CA_Size: Measured size from caliper (Region[180-600] Size [BP])
  • CA_Size_Adpt: Measured size from caliper (Region[120-140] Size [BP])
  • CA_Molarity: Measured molarity from caliper (Region[180-600] Molarity (nmol/l)). NOTE! This conflicts with the existing annotation that is the calculated value but this should be stored in a new annotation instead.
  • CA_Molarity_Adpt: Measured molarity from caliper (Region[120-140] Molarity (nmol/l)).
  • Library_Size_Est: Weighted size
  • Library_Molarity_Est: Calculated molarity (the old CA_Molarity annotation)
  • Library_Frac_Adpt: Fraction of adapter molecules in percent.

This information will then be used in the pooling wizard to detect problems. See other ticket #597.

Change History (6)

comment:1 Changed 6 years ago by Nicklas Nordborg

Description: modified (diff)

comment:2 Changed 6 years ago by Nicklas Nordborg

Status: newassigned

comment:3 Changed 6 years ago by Nicklas Nordborg

(In [2447]) References #596: Import adapter peak information when registering libraries

Replaced 'CA_Molarity' with 'Library_Molarity_Est'. No changes to importer.

comment:4 Changed 6 years ago by Nicklas Nordborg

(In [2448]) References #596: Import adapter peak information when registering libraries

Added all CA_* annotations and importing them from the well table file. Calculations are still done the old way (not weighted).

comment:5 Changed 6 years ago by Nicklas Nordborg

(In [2449]) References #596: Import adapter peak information when registering libraries

Added remaining annotations (calculated values) and implemented the calculations.

comment:6 Changed 6 years ago by Nicklas Nordborg

Resolution: fixed
Status: assignedclosed
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