# Count feature barcodes * [What is Count feature barcodes?](#what-is-count-feature-barcodes) * [Running Count feature barcodes](#running-count-feature-barcodes) * [How does Count feature barcodes work?](#how-does-count-feature-barcodes-work) ## What is Count feature barcodes? *Count feature barcodes* is a tool for quantifying the number of feature barcodes per cell from CITE-Seq, cell hashing, or other feature barcoding assays to measure protein expression. The input for *Count feature barcodes* is FASTQ files. ## Running Count feature barcodes *Count feature barcodes* will run on any unaligned reads data node. * Click the data node containing your unaligned reads containing feature barcodes * Click the **Quantification** section in the toolbox * Click **Count feature barcodes** The task set up page allows you to configure the settings for your assay (Figure 1).
Figure 1. Count feature barcodes task set up page
* Choose the *Prep kit* from the drop-down menu For more details on adding *Prep kits*, please see our documentation on [Trim tags](https://help.connected.illumina.com/partek/partek-flow/user-manual/task-menu/pre-alignment-tools/trim-tags). The prep kit should include cell barcode and unique molecular identifiers (UMIs) locations. * Check *Map feature barcodes* box (optional) This is only necessary for processing data from 10X Genomics' Feature Barcoding assay (v3+ chemistry), which utilizes BioLegend TotalSeq-B. For all other assays, leave this box unchecked. * Choose the *Barcode location* For BioLegend TotalSeq-A, choose **bases 1-15**. For BioLegend TotalSeq-B/C, choose **bases 11-25**. For other locations, select **Custom** and specify the start and stop positions. * Choose a *Sequences* text file This tab-delimited text file should have the feature ID in the first column and the nucleotide sequence in the second column. Do not include column headers. See Figure 2 for an example. ![Figure 2. Example of how the sequences tab-delimited text (.txt) file should be formatted](https://1384254481-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FJVEESmJAPppJ3ijFq5aR%2Fuploads%2Fgit-blob-b801b5f5bc78c9e18edf1d55c051ee1ababe4d34%2FPartek_Flow_Counts_feature_barcodes_sequence_file_example.png?alt=media) * Check *Keep bam files* box (optional) This option will retain the alignment BAM files instead of automatically deleting them when the task is complete. An extra *Aligned reads* output data node will be produced on the task graph. This option is unchecked by default to save on disk space. * Click **Finish** to run The output of *Count feature barcodes* is a *Single cell counts* data node. ## How does Count feature barcodes work? *Count feature barcodes* uses a series of tasks available independently in Partek Flow to process the input FASTQ files. The output files generated by these tasks are not retained in the *Count feature barcodes* output, with the exception of BAM files if *Keep bam files* is checked. [Trim tags](https://help.connected.illumina.com/partek/partek-flow/user-manual/task-menu/pre-alignment-tools/trim-tags) identifies the UMI and cell barcode sequences. The *Prep kit* is specified using the *Prep kit* setting. [Trim bases](https://help.connected.illumina.com/partek/partek-flow/user-manual/task-menu/pre-alignment-tools/trim-bases) trims the insert read to include only the feature barcode sequence. *Trim bases* is set to **Both ends** for *Trim based on* with the start and stop set by the *Barcode location* preference and the *Min read length* set to **1**. [Bowtie](https://help.connected.illumina.com/partek/partek-flow/user-manual/task-menu/aligners) is used to align the reads to the sequences specified in the *Sequences* text file. *Bowtie* is set to **Ignore quality limit** for the *Alignment mode*. Other settings are default. [Deduplicate UMIs](https://help.connected.illumina.com/partek/partek-flow/user-manual/task-menu/post-alignment-tools/deduplicate-umis) consolidates duplicate reads based on UMIs. *Deduplicate UMIs* is set to **Retain only one alignment per UMI**. [Filter barcodes](https://help.connected.illumina.com/partek/partek-flow/user-manual/task-menu/filtering/filter-barcodes) filters the cell barcodes to include cells and not empty droplets. *Filter barcodes* is set to **Automatic**. Quantify barcodes counts the number of UMIs per cell for each feature in the *Sequences* file. *Quantify barcodes* uses default settings. To perform these steps individually instead of using the *Count feature barcodes* task, you will need to generate a FASTA and GTF file containing the feature barcode IDs and sequences instead of a text file and build an index file for the Bowtie aligner. For more information about library file management, please see [Library File Management](https://help.connected.illumina.com/partek/partek-flow/user-manual/settings/components/library-file-management). ## Additional Assistance If you need additional assistance, please visit [our support page](http://www.partek.com/support) to submit a help ticket or find phone numbers for regional support.