Error "Can't open NA.2bit to read"

[sejjbia]

Hi
I am trying to run the test on the fastq files contained in the validationDataSet/Data folder following your instructions but I get the following error after processing of clone1-4

(...)
sample clone1-4
barcoded 104
LTRed 92
linkered 102
ltredlinkered 90
[1] TRUE
mustOpen: Can't open NA.2bit to read: No such file or directory
[1] TRUE
Error in strsplit(alignFile, "/")[[1]] : subscript out of bounds
Calls: alignSeqs
Execution halted

processingParams.tsv are as follows

qualityThreshold badQualityBases qualitySlidingWindow mingDNA minPctIdent maxAlignStart maxFragLength refGenome
? 5 10 30 95 5 2500 hg18

Genome hg18 is installed properly and hg18.2bit is generated as well
(tried also changing to hg19. Same error)
Can you help?

[helixscript]

Hi Sejjbia, Sorry for the trouble with the software. This is an older software project and we are currently working to make a more general and exportable version. First, I am assuming that you are using the deployment branch from GitHub repository, have successfully built the Conda environment, and are executing from within it. The presence of hg18.2bit proves that BS genome libraries are functioning. The refGenome value is typically taken from the sampleInfo file not the processing parameter file. Please add a column to your sampleInfo file 'refGenome' and set this value to 'hg18' and let me know if this resolves the issue. Best, John

…

On Mon, Nov 4, 2019 at 4:42 PM sejjbia ***@***.***> wrote: Hi I am trying to run the test on the fastq files contained in the validationDataSet/Data folder following your instructions but I get the following error after processing of clone1-4 (...) sample clone1-4 barcoded 104 LTRed 92 linkered 102 ltredlinkered 90 [1] TRUE *mustOpen: Can't open NA.2bit to read: No such file or directory* [1] TRUE *Error in strsplit(alignFile, "/")[[1]] : subscript out of bounds Calls: alignSeqs Execution halted* processingParams.tsv are as follows qualityThreshold badQualityBases qualitySlidingWindow mingDNA minPctIdent maxAlignStart maxFragLength refGenome ? 5 10 30 95 5 2500 hg18 Genome hg18 is installed properly and hg18.2bit is generated as well (tried also changing to hg19. Same error) Can you help? — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#75?email_source=notifications&email_token=AB2ISQ3SQFUDJGM7EVNZ2BDQSCJKXA5CNFSM4JI2LTNKYY3PNVWWK3TUL52HS4DFUVEXG43VMWVGG33NNVSW45C7NFSM4HWXG3HQ>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AB2ISQ4RBNI2VTVNFTBOXVLQSCJKXANCNFSM4JI2LTNA> .

-- John K. Everett http://www.linkedin.com/in/JohnEverettPhD

[sejjbia]

First, I am assuming that you are using the deployment branch from GitHub YES
have successfully built the Conda environment, and are executing from within it. YES
Please add a column to your sampleInfo file 'refGenome' and set this value to 'hg18' and let me know I tried your fix but unfortunately it's not working. I still get the same error.

This is the content of the sampleInfo.tsv file
alias linkerSequence bcSeq gender primer ltrBit largeLTRFrag vectorSeq refGenome clone1-1 CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT AATCCGTACAGC f GAAAATC TCTAGCA TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT p746vector.fasta hg18 clone1-2 CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT ACACCTGGTGAT f GAAAATC TCTAGCA TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT p746vector.fasta hg18 clone1-3 CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT TATCGTTGACCA f GAAAATC TCTAGCA TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT p746vector.fasta hg18 clone1-4 CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT TTACTGTGCGAT f GAAAATC TCTAGCA TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT p746vector.fasta hg18

This is the whole script call and output I get

~/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet$  Rscript ~/INSPIIRED-INSPIIRED-deployment/components/intSiteCaller/intSiteCaller.R
AATCCGTACAGC 	 Data/demultiplexedReps/clone1-1_R1.fastq.gz 
ACACCTGGTGAT 	 Data/demultiplexedReps/clone1-2_R1.fastq.gz 
TATCGTTGACCA 	 Data/demultiplexedReps/clone1-3_R1.fastq.gz 
TTACTGTGCGAT 	 Data/demultiplexedReps/clone1-4_R1.fastq.gz 
AATCCGTACAGC 	 Data/demultiplexedReps/clone1-1_R2.fastq.gz 
ACACCTGGTGAT 	 Data/demultiplexedReps/clone1-2_R2.fastq.gz 
TATCGTTGACCA 	 Data/demultiplexedReps/clone1-3_R2.fastq.gz 
TTACTGTGCGAT 	 Data/demultiplexedReps/clone1-4_R2.fastq.gz 
[1] TRUE
                     1                                                                                                             
alias                clone1-1                                                                                                      
linkerSequence       CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT                                                               
bcSeq                AATCCGTACAGC                                                                                                  
gender               f                                                                                                             
primer               GAAAATC                                                                                                       
ltrBit               TCTAGCA                                                                                                       
largeLTRFrag         TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT                                                                            
vectorSeq            p746vector.fasta                                                                                              
refGenome            hg18                                                                                                          
linkerCommon         AGTCCCTTAAGCGGAG                                                                                              
qualityThreshold     ?                                                                                                             
badQualityBases      5                                                                                                             
qualitySlidingWindow 10                                                                                                            
mingDNA              30                                                                                                            
minPctIdent          95                                                                                                            
maxAlignStart        5                                                                                                             
maxFragLength        2500                                                                                                          
refGenome            hg18                                                                                                          
read1                /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-1_R1.fastq.gz
read2                /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-1_R2.fastq.gz
         [,1]    
sample   clone1-1
barcoded 87      
              [,1]    
sample        clone1-1
barcoded      87      
LTRed         80      
linkered      85      
ltredlinkered 78      
[1] TRUE
                     2                                                                                                             
alias                clone1-2                                                                                                      
linkerSequence       CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT                                                               
bcSeq                ACACCTGGTGAT                                                                                                  
gender               f                                                                                                             
primer               GAAAATC                                                                                                       
ltrBit               TCTAGCA                                                                                                       
largeLTRFrag         TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT                                                                            
vectorSeq            p746vector.fasta                                                                                              
refGenome            hg18                                                                                                          
linkerCommon         AGTCCCTTAAGCGGAG                                                                                              
qualityThreshold     ?                                                                                                             
badQualityBases      5                                                                                                             
qualitySlidingWindow 10                                                                                                            
mingDNA              30                                                                                                            
minPctIdent          95                                                                                                            
maxAlignStart        5                                                                                                             
maxFragLength        2500                                                                                                          
refGenome            hg18                                                                                                          
read1                /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-2_R1.fastq.gz
read2                /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-2_R2.fastq.gz
         [,1]    
sample   clone1-2
barcoded 73      
              [,1]    
sample        clone1-2
barcoded      73      
LTRed         66      
linkered      72      
ltredlinkered 65      
[1] TRUE
                     3                                                                                                             
alias                clone1-3                                                                                                      
linkerSequence       CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT                                                               
bcSeq                TATCGTTGACCA                                                                                                  
gender               f                                                                                                             
primer               GAAAATC                                                                                                       
ltrBit               TCTAGCA                                                                                                       
largeLTRFrag         TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT                                                                            
vectorSeq            p746vector.fasta                                                                                              
refGenome            hg18                                                                                                          
linkerCommon         AGTCCCTTAAGCGGAG                                                                                              
qualityThreshold     ?                                                                                                             
badQualityBases      5                                                                                                             
qualitySlidingWindow 10                                                                                                            
mingDNA              30                                                                                                            
minPctIdent          95                                                                                                            
maxAlignStart        5                                                                                                             
maxFragLength        2500                                                                                                          
refGenome            hg18                                                                                                          
read1                /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-3_R1.fastq.gz
read2                /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-3_R2.fastq.gz
         [,1]    
sample   clone1-3
barcoded 101     
              [,1]    
sample        clone1-3
barcoded      101     
LTRed         95      
linkered      101     
ltredlinkered 95      
[1] TRUE
                     4                                                                                                             
alias                clone1-4                                                                                                      
linkerSequence       CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT                                                               
bcSeq                TTACTGTGCGAT                                                                                                  
gender               f                                                                                                             
primer               GAAAATC                                                                                                       
ltrBit               TCTAGCA                                                                                                       
largeLTRFrag         TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT                                                                            
vectorSeq            p746vector.fasta                                                                                              
refGenome            hg18                                                                                                          
linkerCommon         AGTCCCTTAAGCGGAG                                                                                              
qualityThreshold     ?                                                                                                             
badQualityBases      5                                                                                                             
qualitySlidingWindow 10                                                                                                            
mingDNA              30                                                                                                            
minPctIdent          95                                                                                                            
maxAlignStart        5                                                                                                             
maxFragLength        2500                                                                                                          
refGenome            hg18                                                                                                          
read1                /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-4_R1.fastq.gz
read2                /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-4_R2.fastq.gz
         [,1]    
sample   clone1-4
barcoded 104     
              [,1]    
sample        clone1-4
barcoded      104     
LTRed         92      
linkered      102     
ltredlinkered 90      
[1] TRUE
mustOpen: Can't open NA.2bit to read: No such file or directory
[1] TRUE
Error in strsplit(alignFile, "/")[[1]] : subscript out of bounds
Calls: alignSeqs
Execution halted

[helixscript]

Hi Sejjbia, Sorry for the trouble. Based on your path, I suspect you are working with Luca Biasco. Please set up a means for me to retrieve your analysis directory including sequencing data and I will take a look on my side. John

…

On Mon, Nov 11, 2019 at 5:17 PM sejjbia ***@***.***> wrote: First, I am assuming that you are using the deployment branch from GitHub *YES* have successfully built the Conda environment, and are executing from within it. *YES* Please add a column to your sampleInfo file 'refGenome' and set this value to 'hg18' and let me know *I tried your fix but unfortunately it's not working. I still get the same error.* *This is the content of the sampleInfo.tsv file* alias linkerSequence bcSeq gender primer ltrBit largeLTRFrag vectorSeq refGenome clone1-1 CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT AATCCGTACAGC f GAAAATC TCTAGCA TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT p746vector.fasta hg18 clone1-2 CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT ACACCTGGTGAT f GAAAATC TCTAGCA TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT p746vector.fasta hg18 clone1-3 CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT TATCGTTGACCA f GAAAATC TCTAGCA TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT p746vector.fasta hg18 clone1-4 CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT TTACTGTGCGAT f GAAAATC TCTAGCA TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT p746vector.fasta hg18 This is the whole script call and output I get ~/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet$ Rscript ~/INSPIIRED-INSPIIRED-deployment/components/intSiteCaller/intSiteCaller.R AATCCGTACAGC Data/demultiplexedReps/clone1-1_R1.fastq.gz ACACCTGGTGAT Data/demultiplexedReps/clone1-2_R1.fastq.gz TATCGTTGACCA Data/demultiplexedReps/clone1-3_R1.fastq.gz TTACTGTGCGAT Data/demultiplexedReps/clone1-4_R1.fastq.gz AATCCGTACAGC Data/demultiplexedReps/clone1-1_R2.fastq.gz ACACCTGGTGAT Data/demultiplexedReps/clone1-2_R2.fastq.gz TATCGTTGACCA Data/demultiplexedReps/clone1-3_R2.fastq.gz TTACTGTGCGAT Data/demultiplexedReps/clone1-4_R2.fastq.gz [1] TRUE 1 alias clone1-1 linkerSequence CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT bcSeq AATCCGTACAGC gender f primer GAAAATC ltrBit TCTAGCA largeLTRFrag TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT vectorSeq p746vector.fasta refGenome hg18 linkerCommon AGTCCCTTAAGCGGAG qualityThreshold ? badQualityBases 5 qualitySlidingWindow 10 mingDNA 30 minPctIdent 95 maxAlignStart 5 maxFragLength 2500 refGenome hg18 read1 /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-1_R1.fastq.gz read2 /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-1_R2.fastq.gz [,1] sample clone1-1 barcoded 87 [,1] sample clone1-1 barcoded 87 LTRed 80 linkered 85 ltredlinkered 78 [1] TRUE 2 alias clone1-2 linkerSequence CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT bcSeq ACACCTGGTGAT gender f primer GAAAATC ltrBit TCTAGCA largeLTRFrag TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT vectorSeq p746vector.fasta refGenome hg18 linkerCommon AGTCCCTTAAGCGGAG qualityThreshold ? badQualityBases 5 qualitySlidingWindow 10 mingDNA 30 minPctIdent 95 maxAlignStart 5 maxFragLength 2500 refGenome hg18 read1 /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-2_R1.fastq.gz read2 /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-2_R2.fastq.gz [,1] sample clone1-2 barcoded 73 [,1] sample clone1-2 barcoded 73 LTRed 66 linkered 72 ltredlinkered 65 [1] TRUE 3 alias clone1-3 linkerSequence CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT bcSeq TATCGTTGACCA gender f primer GAAAATC ltrBit TCTAGCA largeLTRFrag TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT vectorSeq p746vector.fasta refGenome hg18 linkerCommon AGTCCCTTAAGCGGAG qualityThreshold ? badQualityBases 5 qualitySlidingWindow 10 mingDNA 30 minPctIdent 95 maxAlignStart 5 maxFragLength 2500 refGenome hg18 read1 /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-3_R1.fastq.gz read2 /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-3_R2.fastq.gz [,1] sample clone1-3 barcoded 101 [,1] sample clone1-3 barcoded 101 LTRed 95 linkered 101 ltredlinkered 95 [1] TRUE 4 alias clone1-4 linkerSequence CGCAGACATCCCGTCCCATNNNNNNNNNNNNCTCCGCTTAAGGGACT bcSeq TTACTGTGCGAT gender f primer GAAAATC ltrBit TCTAGCA largeLTRFrag TGCTAGAGATTTTCCACACTGACTAAAAGGGTCT vectorSeq p746vector.fasta refGenome hg18 linkerCommon AGTCCCTTAAGCGGAG qualityThreshold ? badQualityBases 5 qualitySlidingWindow 10 mingDNA 30 minPctIdent 95 maxAlignStart 5 maxFragLength 2500 refGenome hg18 read1 /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-4_R1.fastq.gz read2 /home/sejjbia/INSPIIRED-INSPIIRED-deployment/inputs/validationDataSet/Data/demultiplexedReps/clone1-4_R2.fastq.gz [,1] sample clone1-4 barcoded 104 [,1] sample clone1-4 barcoded 104 LTRed 92 linkered 102 ltredlinkered 90 [1] TRUE mustOpen: Can't open NA.2bit to read: No such file or directory [1] TRUE Error in strsplit(alignFile, "/")[[1]] : subscript out of bounds Calls: alignSeqs Execution halted — You are receiving this because you commented. 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