Nov 03, 2021
Version 1
Evaluating large spontaneous deletions in a bovine cell line selected for bovine viral diarrhea virus resistance - MDBK reads mapping to regions in genome not shared with CRIB cell line V.1
- Aspen M Workman1,
- Michael Heaton2,
- Dennis A. Webster3,
- Gregory P Harhay1,
- Tim Smith2,
- Shollie M Falkenberg4,
- Daniel F. Carlson3,
- Tad S Sonstegard3,
- ted.kalbfleisch5
- 1United States Department of Agriculture (USDA) Agricultural Research Service (ARS), US Meat Animal Research Center (USMARC), State Spur 18D, Clay Center, NE 68933, USA;
- 2US Meat Animal Research Center, USDA, Agricultural Research Service, Clay Center, NE, 68901, USA;
- 3Recombinetics Inc., Eagan, MN, 55121, USA;
- 4National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, 50010, USA;
- 5Department of Veterinary Science, Gluck Equine Research Center, University of Kentucky, Lexington, KY, 40506, USA
- USMARC Bacterial Systems Biology Working Group
Protocol Citation: Aspen M Workman, Michael Heaton, Dennis A. Webster, Gregory P Harhay, Tim Smith, Shollie M Falkenberg, Daniel F. Carlson, Tad S Sonstegard, ted.kalbfleisch 2021. Evaluating large spontaneous deletions in a bovine cell line selected for bovine viral diarrhea virus resistance - MDBK reads mapping to regions in genome not shared with CRIB cell line . protocols.io https://dx.doi.org/10.17504/protocols.io.bvv3n68n
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 16, 2021
Last Modified: November 03, 2021
Protocol Integer ID: 50843
Abstract
Bovine viral diarrhea virus (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genesPTPN12,GRID2, andRABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss ofPTPN12,GRID2, orRABGAP1Lgene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.
Inputs
Inputs
Required software
Get RepeatMasked ARS-UCSC2.1 RepeatMasked Bovine Genome features
Select "Cow" genome and ARS-UCSD 2.1 bovine genome, select "Variation and Repeats" from group and RepeatMasker Track and set other parameters as defined in screenshot above, including outputting to file named ARS-UCD1.2.RepeatMask.bed. Click "get output"
Create BED record "Whole Gene" , the click "get BED"
BED file of RepeatMasked Regions of ARS-UCSD bovine genome is 
ARS-UCD1.2.RepeatMask.bed
ARS-UCD1.2.RepeatMask.bed Check BAM Files
Check BAM Files
Cell line library BAM files
Illumina libraries of CRIB and MDBK cell lines
The library names are
- LIB14393_Bovine_CRIBcells.Bt_ARS-UCD1.2.realigned.bam
- LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.bam
check files
Command
Check MDBK BAM files (OS X)
Expected result
367878117 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
1572651 + 0 supplementary
14182468 + 0 duplicates
363791869 + 0 mapped (98.89% : N/A)
366305466 + 0 paired in sequencing
183152733 + 0 read1
183152733 + 0 read2
348244352 + 0 properly paired (95.07% : N/A)
359573946 + 0 with itself and mate mapped
2645272 + 0 singletons (0.72% : N/A)
10766072 + 0 with mate mapped to a different chr
5380851 + 0 with mate mapped to a different chr (mapQ>=5)
Note
BAM file is acceptable
Command
Check CRIB
Expected result
478473382 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
1983826 + 0 supplementary
17840954 + 0 duplicates
469694793 + 0 mapped (98.17% : N/A)
476489556 + 0 paired in sequencing
238244778 + 0 read1
238244778 + 0 read2
451693766 + 0 properly paired (94.80% : N/A)
464266014 + 0 with itself and mate mapped
3444953 + 0 singletons (0.72% : N/A)
11903898 + 0 with mate mapped to a different chr
5382690 + 0 with mate mapped to a different chr (mapQ>=5)
Note
BAM file is acceptable
Data Conversion
Data Conversion
Convert BAM to BED
Command
Convert BAM to BED
Command
Convert BAM to BED
Groom Data
Groom Data
Remove features from the MDBK file that intersect with repeats in ARS-UCD1.2.RepeatMask.bed
Command
Find those features (reads) present in MDBK that do no overlap features (reads) in CRIB
Command
Find reads in MDBK mapping to genome absent in CRIB
Output: MDBK+_CRIB-.bed
MDBK+_CRIB-.bed