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  1. IGB
  2. IGBF-3838

Obtain and view sequence read alignments for PBMC dataset from Seurat vignette

        Details

        • Type: Task
        • Status: Closed (View Workflow)
        • Priority: Major
        • Resolution: Done
        • Affects Version/s: None
        • Fix Version/s: None
        • Labels:
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          Description

          For this task, locate a BAM format data files for the RNA-Seq data set described in this Seurat tutorial:

          The above tutorial demonstrates features of the Seurat single-cell RNA-Seq data analysis library using data from peripheral blood mononuclear Cells (PBMC) originally from the 10X Genomics Web site.

          Open the data file in IGB. Use the Seurat library (in R) to locate some genes with high counts and then check that IGB shows the same or similar numbers of read alignments.

          Then, make it possible for other people to also open the file and view the contents in IGB.

          Note: If you get stuck on figuring out how to use Seurat for this, ask for help from one of our many local R experts.

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            2. 2024-08-12-IQSEC1-PBMC-scRNA-Seq.png
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              Ann Loraine

              Issue Links

              relates to

              Task - A task that needs to be done. IGBF-3863 Investigate Seurat marker gene alignments in IGB

              • Major - Major loss of function.
              • Closed

              Task - A task that needs to be done. IGBF-3864 Find more single cell RNA seq datasets in10XGenomics

              • Major - Major loss of function.
              • Closed

                Activity

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                nfreese Nowlan Freese added a comment - - edited

                The 10X data is currently located at: https://www.10xgenomics.com/datasets/3-k-pbm-cs-from-a-healthy-donor-1-standard-1-1-0
                Note that the website will ask for some personal information.

                The bam file is aligned against the hg19 genome (H_sapiens_Feb_2009)

                Documentation for 10X Genomics Cell Ranger Barcoded BAM tags: https://www.10xgenomics.com/support/software/cell-ranger/latest/analysis/outputs/cr-outputs-bam

                Part of the SAM header: 

                STAR	ID:STAR	VN:STAR_2.5.1b	CL:STAR   --runThreadN 4   --genomeDir /mnt/opt/refdata_cellranger/hg19-1.1.0/star   --readFilesIn /mnt/yard/rudy/testing/cellranger_110/public_datasets/pbmc3k/pbmc3k/CELLRANGER_CS/CELLRANGER/EXTRACT_READS/fork0/chnk0/files/reads.fastq/1.fastq      --outStd BAM_Unsorted   --outSAMtype BAM   Unsorted      --outSAMunmapped Within      --chimOutType WithinBAM   --quantMode TranscriptomeSAM      --quantTranscriptomeBan Singleend
                Show
                nfreese Nowlan Freese added a comment - - edited The 10X data is currently located at: https://www.10xgenomics.com/datasets/3-k-pbm-cs-from-a-healthy-donor-1-standard-1-1-0 Note that the website will ask for some personal information. The bam file is aligned against the hg19 genome (H_sapiens_Feb_2009) Documentation for 10X Genomics Cell Ranger Barcoded BAM tags: https://www.10xgenomics.com/support/software/cell-ranger/latest/analysis/outputs/cr-outputs-bam Part of the SAM header: STAR ID:STAR VN:STAR_2.5.1b CL:STAR --runThreadN 4 --genomeDir /mnt/opt/refdata_cellranger/hg19-1.1.0/star --readFilesIn /mnt/yard/rudy/testing/cellranger_110/public_datasets/pbmc3k/pbmc3k/CELLRANGER_CS/CELLRANGER/EXTRACT_READS/fork0/chnk0/files/reads.fastq/1.fastq --outStd BAM_Unsorted --outSAMtype BAM Unsorted --outSAMunmapped Within --chimOutType WithinBAM --quantMode TranscriptomeSAM --quantTranscriptomeBan Singleend
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                ann.loraine Ann Loraine added a comment - - edited

                Review from Ann Loraine:

                I looked IQSEC1. The alignments are really weird looking! Please see attached image. I see what appears to be a pretty significant problem in that it really seems like lots and lots of the alignments are not matching the exon/intron boundaries of the gene models.

                Request for Karthik Raveendran:

                Is there any on-line documentation for this alignment file? Can you provide some links so that we can investigate further? If this is truly the source of the big matrix of counts, then there is a very serious problem here that might blow up the entire field. Unlikely! But we need to figure out why the alignments do not appear match up with the gene models very well, regardless.

                To complete this task, please add relevant links and pass the ticket back to "Needs first level review." We'll then make a new task to investigate why the alignments do not match the gene models very well.

                Show
                ann.loraine Ann Loraine added a comment - - edited Review from Ann Loraine : I looked IQSEC1. The alignments are really weird looking! Please see attached image. I see what appears to be a pretty significant problem in that it really seems like lots and lots of the alignments are not matching the exon/intron boundaries of the gene models. Request for Karthik Raveendran : Is there any on-line documentation for this alignment file? Can you provide some links so that we can investigate further? If this is truly the source of the big matrix of counts, then there is a very serious problem here that might blow up the entire field. Unlikely! But we need to figure out why the alignments do not appear match up with the gene models very well, regardless. To complete this task, please add relevant links and pass the ticket back to "Needs first level review." We'll then make a new task to investigate why the alignments do not match the gene models very well.
                Hide
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                karthik Karthik Raveendran added a comment -

                The following genes (with high counts in count matrix file) was observed:
                IQSEC1
                TCEANC
                DNASE1L1
                MLLT3
                GDPD5
                SNHG9
                CPNE7
                FTL
                RPL13A

                No match found for the following:
                TCEB3
                AP003733.1
                AP000769.7
                TRIM69

                No reads for the following:
                B3GALT4

                Show
                karthik Karthik Raveendran added a comment - The following genes (with high counts in count matrix file) was observed: IQSEC1 TCEANC DNASE1L1 MLLT3 GDPD5 SNHG9 CPNE7 FTL RPL13A No match found for the following: TCEB3 AP003733.1 AP000769.7 TRIM69 No reads for the following: B3GALT4
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                nfreese Nowlan Freese added a comment -

                Karthik Raveendran - I attached the quickload pointing at the data 10X_PBMC3k files online. Try downloading and adding the quickload to IGB and make sure the data load and look the same as the bam file you downloaded.

                Show
                nfreese Nowlan Freese added a comment - Karthik Raveendran - I attached the quickload pointing at the data 10X_PBMC3k files online. Try downloading and adding the quickload to IGB and make sure the data load and look the same as the bam file you downloaded.
                Hide
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                karthik Karthik Raveendran added a comment -

                Finding some genes with high UMI counts for a cell was done but to check that IGB shows the same number of reads alignments of a gene from a particular cell is a challenge. After discussing with Nowlan Freese on Wednesday, working on filter by or color by function where a cell can be filtered or colored would make it easy to finish the task

                Show
                karthik Karthik Raveendran added a comment - Finding some genes with high UMI counts for a cell was done but to check that IGB shows the same number of reads alignments of a gene from a particular cell is a challenge. After discussing with Nowlan Freese on Wednesday, working on filter by or color by function where a cell can be filtered or colored would make it easy to finish the task

                  People

                  • Assignee:
                    karthik Karthik Raveendran
                    Reporter:
                    ann.loraine Ann Loraine
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                    • Created:
                      Updated:
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