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
Aspen M Workman, Michael Heaton, Dennis A. Webster, Gregory P Harhay, Tim Smith, Shollie M Falkenberg, Daniel F. Carlson, Tad S Sonstegard, ted.kalbfleisch
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 genes PTPN12 , GRID2 , and RABGAP1L . 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 of PTPN12 , GRID2 , or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.
Steps
Inputs
Required software
Software
| Value | Label |
|---|---|
| samtools | NAME |
| GitHub | REPOSITORY |
| http://htslib.org/ | DEVELOPER |
| https://github.com/samtools/samtools/releases/tag/1.9 | LINK |
| 1.9 | VERSION |
Software
| Value | Label |
|---|---|
| Bedtools | NAME |
| https://github.com/arq5x/bedtools2 | REPOSITORY |
| http://quinlanlab.org | DEVELOPER |
| https://bedtools.readthedocs.io/en/latest/ | LINK |
| 2.27.1 | VERSION |
Get RepeatMasked ARS-UCSC2.1 RepeatMasked Bovine Genome features
Goto http://genome.ucsc.edu/cgi-bin/hgTables to create RepeatMasked ARS-UCSD2.1 bovine genome
<img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.bvv3n68n/Screen%20Shot%202021-06-16%20at%201.48.45%20PM.png" alt="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"" loading="lazy" title="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""/>
<img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.bvv3n68n/Screen%20Shot%202021-06-16%20at%201.47.24%20PM.png" alt="Create BED record "Whole Gene" , the click "get BED"" loading="lazy" title="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
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
LIB14393_Bovine_CRIBcells.Bt_ARS-UCD1.2.realigned.bam
#Check MDBK BAM files (OS X)
samtools flagstat LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.bam
LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.bam
#Check CRIB
samtools flagstat LIB14393_Bovine_CRIBcells.Bt_ARS-UCD1.2.realigned.bam
Data Conversion
Convert BAM to BED
#Convert BAM to BED
bamToBed -i LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.bam > LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.bed
#Convert BAM to BED
bamToBed -i LIB14393_Bovine_CRIBcells.Bt_ARS-UCD1.2.realigned.bam > LIB14393_Bovine_CRIBcells.Bt_ARS-UCD1.2.realigned.bed
Groom Data
Remove features from the MDBK file that intersect with repeats in ARS-UCD1.2.RepeatMask.bed
bedtools intersect -a LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.bed -b ARS-UCD1.2.RepeatMask.bed -v > LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.RepMask.bed
Find those features (reads) present in MDBK that do no overlap features (reads) in CRIB
#Find reads in MDBK mapping to genome absent in CRIB
nohup bedtools intersect -a LIB14394_Bovine_MDBKcells.Bt_ARS-UCD1.2.realigned.RepMask.bed -b LIB14393_Bovine_CRIBcells.Bt_ARS-UCD1.2.realigned.bed -v > MDBK+_CRIB-.bed &
```Output: [MDBK+_CRIB-.bed](https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.bvv3n68n/eagvjrq63.bed)
