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BlueBee Data Analysis Pipeline

Automated data analysis for QuantSeq FWD-UMI libraries is available on the BlueBee® Genomics Platform (Figure 2).
To analyze data from QuantSeq FWD libraries that contain UMIs, simply use the activation code included with your QuantSeq FWD library prep kit and select the respective "FWD-UMI" pipeline when setting up your data analysis run.
Please note that the number of free data analysis runs available with the activation code included with each QuantSeq FWD kit, is equal to the number of reactions in the kit (e.g., you get 96 data analysis runs with a 96 prep QuantSeq FWD Kit (Cat. No. 015.96), meaning you can analyze sequencing data for 96 .fastq files).
Therefore, you should choose either the FWD or FWD-UMI pipeline, depending on how your libraries were prepared. If you wish to run both options, additional Activation Codes will need to be purchased (Cat. No. 090). If you start the wrong pipeline for your data you can abort or stop the run before it is completed without losing your allocated runs. If the run is completed then additional runs will need to be purchased.
QuantSeq FWD libraries prepared from blood using Globin Block (RS-GBHs or RS-GBSs, Cat. No. 070 and 071) should be analyzed using the standard FWD pipeline for data analysis; unless UMIs are also included and then the FWD-UMI pipeline should be selected.

Be sure to only select the "FWD-UMI" pipelines for UMI-containing libraries, otherwise duplicate reads will not be collapsed. NOTE!
Do not run the "FWD-UMI" pipelines for QuantSeq FWD (standard) libraries

that do not contain UMIs. This will incorrectly collapse reads and result in incorrect read count data. Use only the FWD pipelines for standard QuantSeq FWD libraries!
Do not use activation codes provided with the QuantSeq REV Kit (Cat. No. 016) for analysis of FWD-UMI libraries. The UMI Second Strand Synthesis Module is not compatible with the QuantSeq REV Kit.
ATTENTION: Data Analysis access is only free using the provided code, for .fastq input files up to 1.5 GB in size. Larger .fastq file sizes can only be processed using activation codes for large file sizes, which can be purchased additionally (Cat. No. 093 for FWD).
As an alternativeFor further details see Can I analyze my QuantSeq data using the Data Analysis Pipelines on the BlueBee® Genomics Platform?

UMI-Tools

As an alternative to BlueBee, we recommend utilizing the publicly available UMI-Tools package available on GitHub here. Detailed documentation can be found in the ReadTheDocs. The following command line will extract the UMI sequence from the read while removing the adjacent 4 nt TATA spacer:

'umi_tools extract –extract-method=regex –bc-pattern "(?P<umi_1>.{6})(?P<discard_1>TATA).*" -L "/path/to/my_outputlog.txt" -I "/path/to/my_input.fastq.gz" -S "/path/to/my_output.fastq.gz"' 

After alignment, reads can be deduplicated with the following command:

umi_tools dedup -I example.bam –output-stats=deduplicated -S deduplicated.bam 

The deduplication method of UMI-Tools has been published here.

NOTE: The current implementation of this method can take some time and can consume significant memory. If you experience issues with run time or memory usage, please refer to these FAQs.

If you would like to run the package in a less complex way, you can set the parameter:

 "-method=unique"

...

This will only collapse UMIs having identical sequences.

For further information , please refer to our QuantSeq UMI FAQ section here or contact us at support@lexogen.com.
Image Removed Figure 2 | QuantSeq FWD-UMI Data Analysis Pipeline available on the BlueBee® Genomics Platform.