Enhanced Protein Isoform Characterization Through Long-Read Proteogenomics - Workflow Results

<pre> </pre> The detection of physiologically relevant protein isoforms encoded by the human genome is critical to biomedicine. Mass spectrometry (MS)-based proteomics is the preeminent method for protein detection, but isoform-resolved proteomic analysis relies on accurate reference databases that match the sample; neither a subset nor a superset database is ideal. Long-read RNA sequencing (e.g. PacBio, Oxford Nanopore) provides full-length transcript sequencing, which can be used to predict full-length proteins. Here, we describe a long-read proteogenomics approach for integrating matched long-read RNA-seq and MS-based proteomics data to enhance isoform characterization. We introduce a classification scheme for protein isoforms, discover novel protein isoforms, and present the first protein inference algorithm for the direct incorporation of long-read transcriptome data in protein inference to enable detection of protein isoforms that are intractable to MS detection. We have released an open-source Nextflow pipeline that integrates long-read sequencing in a proteomic workflow for isoform-resolved analysis. Companion Repositories: Long-Read-Proteogenomics Workflow GitHub Repository Release Long-Read-Proteogenomics Analysis GitHub Repository Release Companion Datasets Long-Read-Proteogenomics Workflow Sample and Reference Data TEST Data for Long-Read-Proteogenomics Workflow GitHub Actions This Repository contains the complete output from the execution of the Long-Read-Proteogenomics Workflow, using the input from Jurkat Samples and Reference Data. The file <em>jurkat.flnc.bam </em>was 6.5 GB had to be split into 13 separate files and for use should be rejoined -- here are the steps that were used to split the file up. 1. Convert <em>jurkat.flnc.bam</em> (binary format) to sam file (text format) without header: <em>samtools view jurkat.flnc.bam &gt; jurkat.flnc.sam</em> 2. Capture the header: <em>samtools view -H jurkat.flnc.bam &gt; jurkat.flnc.header.sam</em> 3. Split <em>jurkat.flnc.sam</em> into smaller files (aim to get final size under 2GB): <em>split -l 400000 jurkat.flnc.sam jurkat.flnc.chunk.</em> 4. Convert each of these files back to bam for uploading: <em>samtools view -b jurkat.flnc.chunk.a* -o jurkat.flnc.chunk.a*.bam (*=a,b,c,d,e,f,g,h,i,j,k,l,m)</em> After downloading, reverse this process including using the header file which is found in the LRPG-Manuscript-Results-results-results-jurkat-isoseq3-companion-files.tar.gz file&gt; 1. Convert the bam files back to sam files: <em>samtools view jurkat.flnc.chunk.a*.bam &gt; jurkat.flnc.chunk.a*.sam (*=a,b,c,d,e,f,g,h,i,j,k,l,m)</em> 2. Combine the header together with the sam files: <em>cat jurkat.flnc.chunk.a*sam &gt; jurkcat.flnc.sam (</em>verified the same number of lines of the sam files is identical to the number of lines of the original without header: 4,956,761. Header file is 13 lines. 3. Convert to bam files if desired: <em>samtools view -b jurkat.flnc.sam -o jurkat.flnc.bam</em> 4. Rehead with the header file: <em>samtools reheader -P -i jurkat.flnc.header.sam jurkat.flnc.bam</em>