New workflow for ONT, consisting of prep and sequencing

[amandaraud]

We are going to start sequencing samples with ONT for the 100 genomes project, which is planned to start in December. Luckily enough, NGI have been running ONT samples for some time, and already have a LIMS workflow set up for it:

A meeting is planned in order to discuss how much of their implementation can be used by us, and what changes that are needed.

The current deadline for having this in production is around the 5-10th of December, but this might change depending on how the lab and IT implementations progress.

[Karl-Svard]

NGI workflow set-up: https://docs.google.com/spreadsheets/d/1JKTgZmP6pnPfjUJtSt6gL-yTqmHN6PJ8Yp0joG3U_nY/edit#gid=183845737

Comments and requests from LF: https://docs.google.com/document/d/1OQBI0tdQVhBlzRhnlmuUfw4YbKi6uEfgXjpJAuHX-MY/edit

(tell me if you need access to view them)

[Karl-Svard]

The ONT workflow has now been deployed to the production environment

New workflow: ONT

Initial QC (ONT)

• Reception Control (ONT), Reception Control (ONT)
• Qubit QC (DNA) (ONT), Qubit QC (DNA) (ONT)
• Quantit QC (DNA) (ONT), Quantit QC (DNA) (ONT)
• Tapestation Reception Control (ONT), Tapestation Reception Control (ONT)
• Aggregate QC (DNA) (ONT), Aggregate QC (DNA) (ONT)

ONT Library Prep

• ONT Pre-Prep Sort, ONT Pre-Prep Sort
• DNA Fragmentation (ONT), DNA Fragmentation (ONT)
• DNA Size Selection (ONT), DNA Size Selection (ONT)
• Aliquot Samples for ONT Prep, Aliquot Samples for ONT Prep
• ONT End Repair, ONT End Repair
• ONT Adapter Ligation, ONT Adapter Ligation

Library Validation (ONT)

• Qubit QC (Library Validation) (ONT), Qubit QC (Library Validation) (ONT)
• Quantit QC (Library Validation) (ONT), Quantit QC (Library Validation) (ONT)
• Aggregate QC (Library Validation) (ONT), Aggregate QC (Library Validation) (ONT)

ONT Sequencing

• ONT Start Sequencing, ONT Start Sequencing
• ONT Sequencing and Reloading, ONT Sequencing and Reloading
• ONT Finish Sequencing, ONT Finish Sequencing

Delivery v1

• Delivery, Delivery v1

New EPPs created:

Set Date

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy process-to-sample -sudf 'Received at' -pudf 'Date arrived at Clinical Genomics'"

• Reception Control (ONT)

Check Required - Aggregate QC (DNA) (ONT)

bash -c -l "conda activate cg_lims &&  lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf check check-artifact-udfs  -i -au 'Concentration'  -au 'Amount (ng)'"

• Aggregate QC (DNA) (ONT)

Calculate Amount for 50 μl of the samples

bash -c -l "conda activate cg_lims &&  lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf calculate calculate-amount-ng-fmol -n 'DNA Fragmentation (ONT)' --concentration-udf 'Concentration' --volume-udf 'Volume (ul)' --size-udf 'Size (bp)' --preset-volume 50"

• DNA Fragmentation (ONT)

Calculate ONT Aliquot Volumes for fragmentation

bash -c -l "conda activate cg_lims &&  lims -c  /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid}   udf calculate ont-aliquot-volume -n 'DNA Fragmentation (ONT)' --concentration-udf 'Input Concentration (ng/ul)' --size-udf 'Size (bp)' --volume-udf 'Sample Volume (ul)' --buffer-udf 'Volume H2O (ul)' --total-volume-udf 'Total Volume (ul)'"

• DNA Fragmentation (ONT)

Copy UDFs from Reception Control QC

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy artifact-to-artifact --sample-artifact -au 'Input Size (bp)' -sau 'Size (bp)' -au 'Input Amount (ng)' -sau 'Amount (ng)' -au 'Volume (ul)' -sau 'Volume (ul)' -sau 'Concentration' -au 'Input Concentration (ng/ul)'"

• DNA Fragmentation (ONT)
• DNA Size Selection (ONT)

Calculate Aliquotation Volumes for Size Selection

bash -c -l "conda activate cg_lims &&  lims -c  /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid}   udf calculate ont-aliquot-volume -n 'DNA Fragmentation (ONT)' -n 'DNA Size Selection (ONT)' --concentration-udf 'Input Concentration (ng/ul)' --size-udf 'Size (bp)' --volume-udf 'Sample Volume (ul)' --buffer-udf  'Volume H2O (ul)' --total-volume-udf 'Total Volume (ul)' "

• DNA Size Selection (ONT)

Calculate Amount for 37 μl of the samples

bash -c -l "conda activate cg_lims &&  lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf calculate calculate-amount-ng-fmol -n 'DNA Fragmentation (ONT)' -n 'DNA Size Selection (ONT)' --concentration-udf 'Concentration' --volume-udf 'Volume (ul)' --size-udf 'Size (bp)' --preset-volume 37"

• DNA Size Selection (ONT)

Copy UDFs from DNA Fragmentation

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy artifact-to-artifact -n 'DNA Fragmentation (ONT)' -au 'Input Size (bp)' -sau 'Size (bp)' -au 'Input Amount (ng)' -sau 'Amount (ng)' -au 'Volume (ul)' -sau 'Volume (ul)' -sau 'Concentration' -au 'Input Concentration (ng/ul)'"

• DNA Size Selection (ONT)

Calculate ONT Aliquot Volumes

bash -c -l "conda activate cg_lims &&  lims -c  /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid}   udf calculate ont-aliquot-volume -n 'DNA Fragmentation (ONT)' -n 'DNA Size Selection (ONT)' --concentration-udf 'Concentration' --size-udf 'Size (bp)' --volume-udf 'Sample Volume (ul)' --buffer-udf 'Volume H2O (ul)' --total-volume-udf 'Total Volume (ul)'"

• Aliquot Samples for ONT Prep

Copy UDFs from Initial QC or Previous Steps

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy artifact-to-artifact --sample-artifact -au  'Concentration' -sau 'Concentration' -au 'Amount (ng)' -sau 'Amount (ng)' && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy artifact-to-artifact -n 'DNA Fragmentation (ONT)' -n 'DNA Size Selection (ONT)' -au  'Concentration' -sau 'Concentration' -au 'Amount (ng)' -sau 'Amount (ng)' --ignore-fail"

• Aliquot Samples for ONT Prep

Copy Volume from Initial QC or Previous Step

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf set updated-sample-volume -sa && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf set updated-sample-volume -n 'DNA Fragmentation (ONT)' -pudf 'Total Volume (ul)'  --subtract-volume 5 --ignore-fail && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf set updated-sample-volume -n 'DNA Size Selection (ONT)' -pudf 'Total Volume (ul)' --subtract-volume 13 --ignore-fail"

• Aliquot Samples for ONT Prep

Calculate Amount for 60 μl of the samples

bash -c -l "conda activate cg_lims &&  lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf calculate calculate-amount-ng-fmol -n 'DNA Fragmentation (ONT)' -n 'DNA Size Selection (ONT)' --concentration-udf 'Concentration (ng/ul)' --volume-udf 'Volume (ul)' --size-udf 'Size (bp)' --preset-volume 60"

• ONT End Repair

Copy Amount (ng) from ONT Aliquotation

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy artifact-to-artifact  --process-types 'Aliquot Samples for ONT Prep' -au 'Input Amount (ng)' -sau 'Amount (ng)'"

• ONT End Repair

Copy Volume from ONT Aliquotation

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf set updated-sample-volume -n 'Aliquot Samples for ONT Prep' -pudf 'Total Volume (ul)'"

• ONT End Repair

Copy Amount (ng) from ONT End Repair

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy artifact-to-artifact  --process-types 'Aliquot Samples for ONT Prep' -au 'Input Amount (ng)' -sau 'Amount (ng)'"

• ONT Adapter Ligation

Calculate Amount for 24 μl of the samples

bash -c -l "conda activate cg_lims &&  lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf calculate calculate-amount-ng-fmol -n 'DNA Fragmentation (ONT)' -n 'DNA Size Selection (ONT)' --concentration-udf 'Concentration' --volume-udf 'Volume (ul)' --size-udf 'Size (bp)' --preset-volume 24 -i"

• Aggregate QC (Library VAlidation) (ONT)

Calculate ONT Sequencing Aliquotation

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf calculate ont-aliquot-volume -n 'DNA Fragmentation (ONT)' -n 'DNA Size Selection (ONT)' --concentration-udf 'Concentration' --size-udf 'Size (bp)' --volume-udf 'Sample Volume (ul)' --buffer-udf 'Volume Buffer (ul)' --total-volume-udf 'Loading Volume (ul)' --amount-fmol-udf 'Loading Amount (fmol)'"

• ONT Start Sequencing

Copy Concentration and Amount from ONT prep

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy artifact-to-artifact -au 'Concentration' -au 'Amount (fmol)' -n 'ONT Adapter Ligation'"

• ONT Start Sequencing

Copy Input Container Name and Well

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf copy container-to-artifact -n 'ONT Adapter Ligation' --well-udf 'Input Container Well' --container-name-udf 'Input Container Name'"

• ONT Start Sequencing

Generate ONT Sample Sheet

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} files create-ont-sample-sheet -f {compoundOutputFileLuid1}"

• ONT Start Sequencing

Set ONT Experiment Name

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf set set-ont-sequencing-settings"

• ONT Start Sequencing

Calculate Available Amount for ONT Reload

bash -c -l "conda activate cg_lims &&  lims -c  /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf calculate ont-available-sequencing-reload"

• ONT Sequencing and Reloading

Calculate ONT Reload Aliquot Volume

bash -c -l "conda activate cg_lims &&  lims -c  /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid} udf calculate ont-aliquot-volume -n 'DNA Fragmentation (ONT)' -n 'DNA Size Selection (ONT)' --concentration-udf 'Concentration' --size-udf 'Size (bp)' --volume-udf 'Sample Volume (ul)' --buffer-udf 'Buffer Volume (ul)' --amount-fmol-udf 'Reload Amount (fmol)' --total-volume-udf 'Reload Volume (ul)' -m"

• ONT Sequencing and Reloading

Next step Remove (Non output step)

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'if (input.hasValue( ::Rerun:: ) && input.::Rerun:: == true) { nextStep = ::REMOVE:: } else { nextStep = ::ADVANCE:: }' -log {compoundOutputFileLuid1}"

• ONT Finish Sequencing

Parse ONT Run Results

bash -c -l "conda activate cg_lims && lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid4} -p {processLuid} files parse-ont-report --root-path /home/gls/nanopore_data"

• ONT Finish Sequencing

Queue Samples for Rerun (ONT)

bash -c -l "conda activate cg_lims &&  lims -c /home/glsai/.genologics.yaml epps -l {compoundOutputFileLuid0} -p {processLuid}  move  rerun-samples -u 'Rerun' -n 'ONT Adapter Ligation' -w 2152 -s 7487 -i"

• ONT Finish Sequencing

New UDFs created:

Derived Sample

• Centrifugal speed (RCF)
• Input Concentration (ng/ul)
• Input Amount (ng)
• Input Size (bp)
• Amount (fmol)
• Input Container Name
• Input Container Well
• ONT Experiment Name
• ONT Flow Cell Position
• QC Pore Count
• Loading Amount (fmol)
• Reads Generated
• Estimated N50
• Estimated Bases
• QC Passed Bases
• QC Failed Bases

Measurement

• Available Amount (fmol)
• Reload Amount (fmol)
• Reload Run Time (hh:mm)

New Container type

• PromethION Flow cell

[henrikstranneheim]

Fantastic 🥇