How to use the package
This package contains a protocol to create library pools. It includes a macro to create the pool and two rules to check the new pool for index unicity and nucleotide distribution.
The rules make use of the i5 and i7 barcode fields that are included.
This package contains the following entities:
- A content type:
- Library pool.
- A macro:
- Make pool: With a single step of type Mix.
- Two rules to check if the mix is balanced, restricted to trigger in protocol run steps of type Mix only:
- Check index unicity in pool: A rule to check if the same index combination is not used multiple times in samples that are pooled together.
- Check index unicity and nucleotide distribution in pool: Extends on the Check index unicity in pool rule. This rule additionally checks if the nucleotides (A, C, G, T) have a balanced distribution, both in overall distribution over all unique index combination in the mix, and distribution per position for each index.
- Three custom fields on Content:
- Barcode – I7: A field of type dynamic choice on the value table DNA index.
- Barcode – I5: A field of type dynamic choice on the value table DNA index.
- Pooled volume per library: A field of type quantity and dimension volume, with default unit µl.
The package contains the fields for the barcodes i5 and i7 which are checked by the rules. These fields need to be available on the content or a parent thereof that is mixed to create the pool.
DNA indexes should be available in the DNA Index Sets module to fill in on the barcode fields.
The rules are configured to only be available in protocol run steps of type Mix. A workflow that has a protocol with a step of type Mix should be created and the rule added.
The rules can be configured to always trigger by disabling the setting to only trigger on specific protocol run steps.
The rule Check index unicity and nucleotide distribution in pool has three variables that can be adjusted to the lab’s needs:
- MIN_PERCENTAGE_PER_NUCLEOTIDE : The overall minimum percentage that each nucleotide should have.
- MIN_PERCENTAGE_POSITION_NUCLEOTIDE: The minimum percentage per base position that each nucleotide should have.
- MAX_PERCENTAGE_POSITION_NUCLEOTIDE: The maximum percentage per base position that each nucleotide should have.
A rule that is not used can be inactivated in the Rules module.
If the percentage at each position should not be checked, either set MIN_PERCENTAGE_POSITION_NUCLEOTIDE to 0 % and MAX_PERCENTAGE_POSITION_NUCLEOTIDE to 100 % or at the bottom of the rule, comment the following lines:
The macro can be adjusted to create a different content type for the pooling and the status can be updated to a different one from the default content status workflow.
The following error messages can be thrown by the rules:
- This error message can be thrown by both rules:
- “Non unique sequences found for i7-i5: ATTACTCG-TATAGCCT for libraries barcode1 and barcode2“: This means that the combination i7-i5 for the two mentioned samples (identified by their barcode, here barcode1 and barcode2) have the same sequence and should therefore not be pooled together. If the samples have been derived, the barcode of the original sample will be shown. This message can be thrown by both rules.
- These error messages can only be thrown by the Check index unicity and nucleotide distribution in pool rule:
- “Only 15% overall for C”: The percentage of C over all the sequences pooled together is below 15%. The default minimum for each nucleotide should be 20% (MIN_PERCENTAGE_PER_NUCLEOTIDE), therefore the samples cannot be pooled together.
- “Less than 12% for C at i5 position: 2”: The percentage of C at position 2 of the i5 index sequences is lower than 12%. The default minimum value for each nucleotide at each position is 12.5% (MIN_PERCENTAGE_POSITION_NUCLEOTIDE), therefore the samples cannot be pooled together.
- “More than 65% for T at i7 position: 3”: The percentage of T at position 2 of the i7 index sequence is higher than 65%. The default maximum value for each nucleotide at each position is 62.5% (MAX_PERCENTAGE_POSITION_NUCLEOTIDE), therefore the samples cannot be pooled together.
Where to Look Next
These references have further information on how to configure or use the package contents after the initial installation and integration.
- Content Management > Content Types
- Miscellaneous > Fields > Custom Fields
- Miscellaneous > Rules > Mix Rules
- Miscellaneous > Macro
This package is also available in combination with a protocol to run the macro in and check the rules. This package can be imported in the Workflow Management module in a workflow by clicking on “Import protocol.” Contact your SLIMS administrator for additional help.