Updating to Reggie 4.23

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IMPORTANT!
This update requires a lot of manual work to install. Some things should be prepared in advance and some things should be done after Reggie 4.23 has been installed. Make sure to assign enough time for the update. No-one should be working with registrations and other things during the upgrade since some wizards may not work correctly until the update has been completed. It is also important to no secondary analysis jobs are running, since this may cause the results to be incorrectly registered or not registered at all.

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1. New pipeline scripts

There are new pipeline scripts: ​http://baseplugins.thep.lu.se/browser/other/pipeline/trunk Download and install at a suitable location on the prime cluster.

2. Changes in reggie-config.xml

There are several changes in reggie-config.xml:

• The path to the pipeline scripts must be changed to point to the new version.
• The path to Picard must be changed to a newer version: /usr/local/packages/picard-tools/2.20.0.
• A new section for the MIPs demux has been added <demux-mips>. The default parameters can probably be used.
• The parameter syntax for Picard has been changed. The old syntax key=value has been changed to -key value. This affects several existing configuration settings:
  • <demux>/<extract-options>
  • <demux>/<fastq-options>
  • <align>/<mark-duplicates-options>
  • <align-hisat>/<mark-duplicates-options>
• It is now possible to give other users access to files created by the secondary analysis for external samples. This is based on the prefix that is used in the names of external RNA/DNA. Add <groupname prefix="...">...</groupname> entries as needed to the <external-samples> section. Make sure that the scanb user that is running the analysis is a member of the specified group.

3. Update BASE to BASE 3.15.2

This version is needed because it includes the Tag batch item importer that is needed in a later stop to import barcodes for the MIPs libraries.

4. Install Reggie 4.23 and run the installation wizard

5. Set a value for the Pipeline annotation on existing items

Almost all existing items from Library and downstream must be given a value for the Pipeline annotation. The table below specifies the value to set for the Pipeline annotation for different types of items. It should be possible to do a manual batch export/import for each row or groups of rows.

Item type	Subtypes	Additional filter	Pipeline value
Extract	Library, PooledLibrary, PooledLibraryAliquot	-	RNAseq
Physical bioassay	FlowCell	-	RNAseq
Derived bioassay	SequencingRun, DemuxedSequences, MergedSequences	-	RNAseq
Derived bioassay	MaskedSequences, AlignedSequences	Job type = HisatAlign	RNAseq/Hisat/StringTie
Derived bioassay	MaskedSequences, AlignedSequences	Job type <> HisatAlign	RNAseq/Legacy
Raw bioassay	Cufflinks	-	RNAseq/Legacy
Raw bioassay	StringTie	-	RNAseq/Hisat/StringTie
Protocol	Demuxing, Merging	-	RNAseq
Software	Demuxing, Merging	-	RNAseq

6. Other updates to existing items

Existing Hardware items of type=Sequencer should be updated with a value for the FlowCellType annotation. It should be either NextSeq or HiSeq.

7. New items that should be created

Item type	Subtypes	Additional settings	Comment
Protocol	DNANormalization	TargetAmount, TargetMinimalAmount, TargetVolume, MIPS_Pool	The protocol to use for normalizing DNA that should go into the MIPs pipeline. If no protocol exists, the option to pre-normalize DNA items (in the DNA/RNA/FlowThrough registration wizard) will not be enabled by default. Thus, it is recommended that a DNANormalization protocol is created only when needed.
Protocol	Library preparation	LibPrepTarget=MIPs	One or more protocols that will appear in the "MIPs library registration" wizard
Protocol	Pooling	LibPrepTarget=MIPs	One or more protocols that will appear in the MIPs pooling wizard
Protocol	Demuxing	Pipeline=MIPs	One or more protocols that will appear in the MIPs demux wizard (demux section)
Software	Demuxing	Pipeline=MIPs	One or more software entries that will appear in the MIPs demux wizard (demux section)
Protocol	Merging	Pipeline=MIPs	One or more protocols that will appear in the MIPs demux wizard (merge section)
Software	Merging	Pipeline=MIPs	One or more software entries that will appear in the MIPs demux wizard (merge section)

8. Barcodes and barcode template plates

Most of the barcode information (such as the sequences, positions, etc.) should be available as a file that can be used with batch importers. Some steps need to be done manually.

1. Import/create Tag items from file using the batch Tag importer and then add annotations with the Annotation importer. The following values are required:

Name	Name of the barcode/tag which must be unique
Pipeline	All items should be set to MIPs
BarcodeSequence	The barcode sequence for the first index read
BarcodeSequence2	The barcode sequence for the second index read

NOTE! The sequences for the second barcode are expected to be in the order that corresponds to the NextSeq sequencer. If the sequencing has been done on a HiSeq they sequences may need to be reverse-complemented before used in the demux. See ​https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/miseq/indexed-sequencing-overview-guide-15057455-05.pdf for more information.

2. A barcode template is represented as a Bioplate item where each coordinate is linked to a Tag item. Start by manually creating 3 bioplates. They should have the following values:

Name	BDC_plate1_v1, BDC_plate2_v1, BDC_plate3_v1
Bioplate type	BarcodeTemplate
Plate geometry	96-well (8x12)
Pipeline (annotation)	MIPs

3. Link the barcodes to templates. This is done by creating (virtual) extracts and link them with tags and positions on one of the bioplates created in the first step. It should be possible to do this with the batch Extract importer:

Name	Name of the extract (recommended to be the same as the name of the Tag)
Tag	Name of the tag
Bioplate	Name of the template bioplate
Biowell row	Row coordinate on the template bioplate (A-H)
Biowell column	Column coordinate on the template bioplate (1-12)

9. User accounts for MIPs pipeline

A user account that should work with the MIPs pipeline need to have the following group/role memberships:

• Groups: SCAN-B Lab, MIPsSecondaryAnalysis
• Roles: User, MIPsLibraryPrep, MIPsPlateDesigner