Basestack Consensus

Parameters

Long Read Run DirectoryDir

Run directory from a MinION, GridION, Mk1C, etc. Must contain several files or folders in it. See manifest, sequencing summary and fastq_pass below

primersText/Dir

Choice of Custom (directory) or pre-loadable options for primer set used (Artic only supported pre-loaded primer set currently)

barcodingText/File

Select barcoding configuration used during demultiplexing. If demultiplexing didn’t take place, any are allowed

basecallingText/File

Select one of many supported basecalling configurations during the Basecalling step (creating fastq files from fast5)

fastq_passDir

Directory of fastq files (can be demultiplexed or not). All fastq files to be analyzed MUST be decompressed (no .gz or .zip format allowed)

manifestList

Contains your ID to barcode mapping in a .tsv (tab-separated) format.

Sequencing SummaryFile

Not inputable. Ensure that it is in the top-level directory (root, same level as the Run Directory). It is required to run some portions. It is output at the end of every Basecalling step from Guppy

Note

Within manifest, one entry must contain the NTC ( case-sensitive, no template control). If you don’t have a NTC, select NB00 as the barcode and NTC as the id.

Returns

Consensuses./artic-pipeline/4-draft-consensus/

  • Complete FASTA files will be output as ...complete.fasta for each barcode

  • This folder and other sibling folders will contain other file formats such as .vcf and .bam for other downstream analysis pipelines.

Report of Run./artic-pipeline/report.pdf

  • Contains important information about your samples (each barcode), lineage information, mutations, etc.

Running Consensus Generation and Reporting

Consensus Generation is the main feature of this application and is used to generate a report of a run directory that was generated from a MinION run. It has multiple steps but is designed to be very automated once a job is submit for analysis.

If you haven’t already done so, download and unzip the Test data folder

Starting a Run

_images/AddRunFolder.png
  1. Select the appropriate test folder first. This folder is either included in the test-data folder in the source of this application OR you can retrieve it within the install location of the app. For example, in C:Program FilesBasestackclientdatatest-data.

    • You can either drag + drop it into the Run Folder field or select it by left clicking and browser to the directory location on your computer

  2. Once a folder has been input, you should see the text fields populate and turn green. If any field is marked as read, it is invalid and should be looked at further for proper formatting. These files should be valid for the test dataset. If you want to use your own data please follow the formatting in run_config.txt, run_info.txt, and manifest.txt. These formats are like:

Note

You may skip this portion if you’d like

  1. run_config.txt

  • This should be 3 rows that dictate the primer (first), basecalling workflow (second), and barcoding cfg (third/last). Separate by tab.

Example:

Target (Unlisted)

Config (Unlisted)

primers

nCov-2019/V3

basecalling

dna_r9.4.1_450bps_hac.cfg

barcoding

barcode_arrs_nb96.cfg

  1. manifest.txt

Example:

Barcode

Sample

NB01

NTC (always required)

NB02

MDHP-00058

NB03

MDHP-00059

.

.

.

.

Note

If you don’t have an NTC (NOT RECOMMENDED EXPERIMENTALLY), set NTC as NB00

  • This should be any number of rows that contain barcode on the left ALWAYS and the sample code on the right. A no-template-control (NTC) must always be specified for a report to be completed. Separate by tab.

  • You are allowed to input your own custom values for each of the 3 files where the app will overwrite that corresponding file on a job submission. That means you can populate these fields by either directly modifying the files OR by inputting them into the input fields

    • These values are currently not validating to their greatest extent so take care to correctly input values and delimit them with tabs if doing this manually.

Lastly, there are three files that are made following a successful sequencing (and basecalling) run. These three are

  1. Sequencing Summary REQUIRED

    • This file is made following basecalling. It contains the mapping and summary stats of all fast5 to fastq generations and must be present in the run directory for report generation

    • If using CLI or stand-alone basecalling you will likely need to move this file from the fastq output savepath to the base run directory.

  2. Throughput….csv OPTIONAL

  3. Drift Correction OPTIONAL

Note

Future updates of Basestack will prevent the job from commencing if the sequencing summary is not present

Starting the process

_images/StartConsensus.png

  1. Once everything is staged, you should see all items update accordingly based on information in the directory.

_images/RunConsensus.png

  1. Hit Start in the upper right-hand corner to start consensus generation.

Note

Depending on your method of installing Docker on Windows, you may receive a notification for docker to share a folder. Hit okay to allow the pipeline to continue. If you run Basestack as an admin, this error will be avoided. You can also opt to share the Basestack folder and sub-folders in the Docker Desktop on Windows as well (see how to do this in the next 2 images)

_images/filesharingoverview.PNG _images/filesharingSelectedFolder.PNG

Note

Simply select the folder that contains the Basestack.exe file by selecting the plus-mark and navigation and selecting it within the browser. In this example it is: …buildwin-unpacked

Checking Logs and Status

_images/LogsConsensus.png

Note

You can see the output of the run in the Log Window container on the bottom of the page. You can also see the Output(s) table begin to change as modules are completed for your run. The final module is the report generation module and should always be 1/1 when complete

Final Report

_images/finalTables.PNG

Note

Once complete, you can view the pdf report by clicking the pdf file icon link underneath the final row’s status of 1/1. You can also traverse to any of the module directories by hitting the link text on the first column for each module. In this example, I’ve chosen Report Generation as my link which is a top-level view of all modules, as well as the report.pdf location. Open this pdf to see your report either from the folder or the pdf link on the left-most column to see your results!