GPL-960 targeted NanoSeq pipeline (like Duplex-seq and ReISC pipelines) [ToR-011]

[rl15]

Description As R&D specialist (Steff L) I would like a targeted NanoSeq pipeline that is heavily based on the Duplex-seq and ReISC pipelines because...

Who the primary contacts are for this work Stef L (RnD)

Stef L wrote (Tuesday, 13 April 2021 at 15:27)

Targeted NanoSeq Story

• Samples enter as currently for Duplex-seq (LDS stock plate and associated manifest). The stock plate could be renamed to LTN stock. • Enable cherrypick functionality in sequencescape. Use the cherrypick functionality that is available in sequencescape. This will enable a cherrypick plate to be created in sequencescape by combining several stock plates. The cherrypick is carried out on the Tecan - use the existing sequencescape bed verification and Tecan GWL file generation functionalities. Output plate LTN Cherrypick. • For samples that don’t need to be cherrypicked the LTN stock plate can be converted to a LTN Cherrypick plate in sequencescape. • The supplied material is sheared by sonication. Upon scanning the LTN Cherrypick plate into Limber enable ‘Add an empty LTN shear plate’ (print labels).

• Bed verification should be available for the Bravo (cherrypick plate on position 7, shear plate onto position 9. • ‘Add an empty LTN Post Shear plate’ (print label).
• Bed verification on Bravo (shear plate onto position 9 and post shear plate onto position 7). • Pipeline now progresses similarly to Duplex-seq pipeline: Create a LTN AL-Lib plate, with this plate creation several labels are printed:
• LTN End repair
• LTN A-Tail
• LTN Ligation
• LTN AL-lib
• LTN AL-lib stamp
• LTN AL-lib QC1
• LTN A-lib QC2 (384 well plate label) • QC2 data is qPCR data that is uploaded to quanthub using the existing Duplex-seq AL-LIB quant type. • A similar qPCR file output is generated as for the duplex-seq pipeline. This is populated by faculty and then re-uploaded in Limber as is the case for the Duplex-seq pipeline. Use the existing template but remove the following columns:
• submit for sequencing
• sub-pool
• coverage • Add a new LTN Dil plate (print barcode).
• The Dil plate also has a hamilton dilution driver file which works exactly as the Duplex-seq driver file (same rules and takes the same information from the previously uploaded file).
• Hamilton bed verification as for Duplex-seq.
• PCR cycle binning: samples need to be binned into different cycles based on upload (as for Duplex-seq) • Add a new LTN PCR XP plate (print barcode) • This plate is then treated like an LCMB plate progressing to ISC. A ReISC type submission will be placed on the LTN PCR XP plate. It then goes down the same process as existing ReISC submission:
• LB Lib PCR-XP (new plate ID for ReISC)
• LB Lib PrePool
• LB Hyb
• LB Cap Lib
• LB Cap Lib PCR
• LB Cap Lib PCR-XP
• LB Cap Lib Pool
• LB Lib Pool Norm. • The final sequencing submission is made by the operational team as is currently the case for ReISC.

[rl15]

Is for CASM Product is also called 'Targeted Duplex-Seq' (See TOR 11) Estimated to be a 12 week project (not clear who estimated)

[SVLensing]

Edit to PCR cycle binning: Before (add new LTN PCR XP plate) insert additional line: create LTN PCR plate and enable (print barcode) and allow index set selection (pWGS). Then create LTN PCR XP Plate (print barcode).

[SVLensing]

Acceptance criteria:

Please see story for all required detail – this is a less detailed summary

A plate containing samples for targeted NanoSeq can arrive from faculty in DNAP. Several plates can potentially be cherrypicked together (not always necessary). The samples can be sheared by sonication by passing through the established shear process (shear plate/post shear plate). Bed verification is enabled on the shearing Bravo as is currently the case and plate label printing is possible. The post shear plate then becomes an AL-Lib plate, and several other labels are printed as the AL-Lib label is printed. qPCR data can be uploaded to quanthub as is currently the case for the Duplex-seq pipeline (Duplex Seq AL-Lib quant type). A similar qPCR output file as for Duplex-seq can then be downloaded but the following columns should be removed: submit for sequencing, sub-pool and coverage. The original Duplex-seq download file should not change as this continues to be required in the established format. A dilution plate should then be created which has the same rules as the Duplex-seq pipeline (e.g. reordering based on PCR cycles). Bed verification is required as for Duplex-seq, a plate label needs to be printed and a Hamilton driver file needs to be generated for the dilution (same rules as for Duplex-seq). A PCR plate is then created with index tag selection. Allow pWGS tag options (A,B,C,D) and plate barcode printing. A PCR XP plate is then created (barcode printing but no bed verification). This PCR XP plate then gets treated as current RelSC submissions. We want to determine which of these samples are to be hybridised with which panel, how they are pooled for hybridisation and how they will later be pooled for sequencing. This data needs to be captured from the customer at this stage. The plate then passes through the existing ReISC pipeline (PCR-XP, PrePool, Hyb, Cap Lib, Cap Lib PCR, Cap Lib PCR-XP, Cap Lib Pool, Lib Pool Norm). The final sequencing submission is then made by the operational team as is currently the case for ReISC.

[stevieing]

For a change I will be the bearer of good news – Manual workaround for GPL-960 looks promising and we will be passing actual samples to evaluate if this is a short term viable option and assess risk. This will exploit the existing Duplex Seq and ReISC LIMS. It’s not ideal as there is a potential for confusion and errors especially as the numbers ramp up but it gives us more time to work through the LIMS and hopefully alleviate some of the pressure. I have copied Stef to keep her in the loop but also to feedback her learning from the mock runs and if their success translates into a leaner/different LIMS ask.

[TWJW-SANGER]

This issue is a duplicate of the issue below, so I am closing this.

913

DPL-183