Micro RNA sequencing

[Nicklas Nordborg]

We need to implement a set of wizard for the MicroRNA sequencing project. The starting point is from Lysate items where a new extraction will made creating RNA extracts with small RNA. The RNA is taken to library preparation and pooled with 64 extracts in a single pool. Sequencing is made with the Illumina NextSeq machine (we already have ~3 runs since before). Demuxing can probably be implemented with Picard using our existing functionality.

This ticket will handle the changes required for lab work, sequencing and demuxing. Alignment and other secondary analysis will be in another ticket (#1607).

Implementation ideas:

• A new subtype sRNA should be defined (it already exists on the production server). It should represent the extracted RNA and be linked to the Lysate. We may need a wizard to import annotations. We probably want the concentration and storage location, etc...
• A new value for the Pipeline annotation should be defined. It could work with sRNAseq? It should be used on items downstream from the sRNA items: Library, Barcode, Pool, FlowCell, SequencingRun and DemuxedSequences. (Note! We could have used the pipeline and regular RNA items instead of sRNA items but there is a lot of existing code that need to be modified to check the pipeline annotation so it is safer to introduce a new subtype)
• We need a wizard to import information about a sequencing run.
  • The 64 libraries that was pooled and their barcodes
  • The flow cell barcode

This should be enough for us to create the Library items and other intermediary items that are needed and it should be possible to start the demux after that.

[Nicklas Nordborg]

In 7806:

References #1606: Micro RNA sequencing

Added sRNA subtype and sRNAseq pipeline. Extraction protocols have been configured to use the pipeline annotation. Existing extraction protocols should be annotated with RNAseq.

[Nicklas Nordborg]

In 7807:

References #1606: Micro RNA sequencing

Updated the extraction wizard to select protocols by pipeline (=RNAseq).

[Nicklas Nordborg]

In 7808:

References #1606: Micro RNA sequencing

Added barcodes for Small RNA seq.

[Nicklas Nordborg]

In 7809:

References #1606: Micro RNA sequencing

Added a section on the Reggie start page with Demux wizards.

[Nicklas Nordborg]

In 7810:

References #1606: Micro RNA sequencing

Started to work on the Start demux wizard.

[Nicklas Nordborg]

In 7811:

References #1606: Micro RNA sequencing

Changed suffix to 'sr' for Small RNA since we already have a lot of items that are registered with that suffix.

[Nicklas Nordborg]

In 7813:

References #1606: Micro RNA sequencing

First version of a demux script for Small RNA. It works and produces FASTQ files, but there is no trimming and other statistics.

[Nicklas Nordborg]

In 7814:

References #1606: Micro RNA sequencing

Changes to the demux script. Removed things that are not going to be used such as fragment size estimation. Implemented a Trimmomatic step for trimming adapters with parameters ILLUMINACLIP:${WD}/srna_adapter.fa:2:30:12 MINLEN:14. Average remaining read lengths are estimated after the trimming (seems to be around 25-30).

[Nicklas Nordborg]

In 7816:

References #1606: Micro RNA sequencing

Changed suffix to back 's' for Small RNA. It was decided that existing items should be renamed.

[Nicklas Nordborg]

In 7818:

References #1606: Micro RNA sequencing

Manual confirmation wizard is implemented and also the auto-confirm except that alignment is not started since it is not yet implemented.

[Nicklas Nordborg]

In 7819:

References #1606: Micro RNA sequencing

Started to implement a registration wizard for sequencing. A sample sheet with all libraries and their barcodes need to be provided and the barcode of the flowcell. Other information will be extracted automatically from the runparameters.xml file from the sequencing.

A lot of validation is still not implemented and neither is the final registration.

[Nicklas Nordborg]

In 7820:

References #1606: Micro RNA sequencing

Implemented validation of aliquot names and barcodes in the sample sheet.

[Nicklas Nordborg]

In 7821:

References #1606: Micro RNA sequencing

Implemented the registration of small RNA sequencing.

[Nicklas Nordborg]

In 7827:

References #1606: Micro RNA sequencing

Added 'mirtrace' to the demux step which generates a QC report (HTML and tab-separated files). We are mainly interested in the fraction or miRNA which is used as a QC parameter to display a warning if it is below 50%.

Mirtrace is run on the FASTQ file before adapter trimming so that it can pick up adapters too. We can't use the generic demux container so instead we create a single container intended for all steps in the sRNA analysis pipeline. There were some issues with picard running out of memory in the IlluminaBasecallsToFastq step but it seems to be solved by modifying the MAX_RECORDS_IN_RAM parameter.

[Nicklas Nordborg]

In 7829:

References #1606: Micro RNA sequencing

Removed _R1 from FASTQ file names since we only have a single read.

[Nicklas Nordborg]

In 7838:

References #1606: Micro RNA sequencing

The MIME type for the Mirtrace HTML report is now set to text/html+javascript so that BASE can enable inline javascripts. This requries at least BASE 3.19.13 (see ​https://base.thep.lu.se/ticket/2327).

[Nicklas Nordborg]

In 7839:

References #1606: Micro RNA sequencing

We need to convert the text/html+javascript MIME type to text/html when downloading files.

[Nicklas Nordborg]

In 7844:

References #1606: Micro RNA sequencing

Use the correct filename (without path) when importing results.