Runnng Demultiplexed Data Files

[tngth...@gmail.com]

Hi Mike,

I ran into problems with your latest AftrRAD.pl script when dealing with de-multiplexed data.

I have a set of uncompressed individual-based fastq files and have them in a folder named “DemultiplexedFiles” in my working directory.

I did not create any barcode or data info files, as it is not required according to your manual.

so I ran this
perl AftrRAD.pl re-GAATTC minDepth-6 minIden-88 dplexedData-1 DataPath-DemultiplexedFiles

Your perl script was interrupted and here’s the output:

Formatting data file 1201.fq
Formatting data file 1202.fq
Formatting data file 1203.fq
Formatting data file 1204.fq
Formatting data file 1205.fq
Formatting data file 2485.fq
Formatting data file 2486.fq
Formatting data file 2487.fq
Formatting data file 2488.fq
Formatting data file 2489.fq
Formatting data file 5033.fq
Formatting data file 5034.fq
Formatting data file 5035.fq
Formatting data file 5036.fq
Formatting data file 5037.fq
Formatting data file 6098.fq
Formatting data file 6099.fq
Formatting data file 6100.fq
Formatting data file 6101.fq
Formatting data file 6102.fq
Formatting data file 6233.fq
Formatting data file 6234.fq
Formatting data file 6235.fq
Formatting data file 6236.fq

Arguments entered are...
minParalog      5
MaxH    90
Phred   33
minDepth        6
dplexedData     1
P2      ATTAGATC
minIden 88
Help    0
stringLength    15
re      GAATTC
DataPath        DemultiplexedFiles
numIndels       3
minQual 20
BarcodePath     Barcodes/

Running AftrRAD...

Recognized 1 data files.

File names are...
 AllSamples.txt 

No such file or directory at AftrRAD.pl line 513.
The barcodes you entered for data file 1 (file AllSamples.txt) are...
 TTTCCG TCTGTT TGCTAC GGCAGC TGATTC GAATAT CGTACG CTTTAC GGGTAT GAATGA TGGGGC GAGCTC ACATTA TGTTAT GGCTAG ACTCAA TCAATG ATGGCG CTCTAT AAATCG CATACG TTAAAA TATCTC GATGCC 

The names you entered for data file 1 (file AllSamples.txt) are...
 1201.fq 1202.fq 1203.fq 1204.fq 1205.fq 2485.fq 2486.fq 2487.fq 2488.fq 2489.fq 5033.fq 5034.fq 5035.fq 5036.fq 5037.fq 6098.fq 6099.fq 6100.fq 6101.fq 6102.fq 6233.fq 6234.fq 6235.fq 6236.fq

I have also tried removing the Data Path option

perl AftrRAD.pl re-GAATTC minDepth-6 minIden-88 dplexedData-1

Formatting data file 1201.fq
Formatting data file 1202.fq
Formatting data file 1203.fq
Formatting data file 1204.fq
Formatting data file 1205.fq
Formatting data file 2485.fq
Formatting data file 2486.fq
Formatting data file 2487.fq
Formatting data file 2488.fq
Formatting data file 2489.fq
Formatting data file 5033.fq
Formatting data file 5034.fq
Formatting data file 5035.fq
Formatting data file 5036.fq
Formatting data file 5037.fq
Formatting data file 6098.fq
Formatting data file 6099.fq
Formatting data file 6100.fq
Formatting data file 6101.fq
Formatting data file 6102.fq
Formatting data file 6233.fq
Formatting data file 6234.fq
Formatting data file 6235.fq
Formatting data file 6236.fq

Arguments entered are...
re      GAATTC
P2      ATTAGATC
minParalog      5
BarcodePath     Barcodes/
stringLength    15
dplexedData     1
DataPath        Data/
numIndels       3
minIden 88
minQual 20
minDepth        6
Phred   33
MaxH    90
Help    0

Running AftrRAD...

Recognized 1 data files.

File names are...
 AllSamples.txt 

Use of uninitialized value $TotalBarcodeNonMatches in concatenation (.) or string at AftrRAD.pl line 957.
Use of uninitialized value $TotalBarcodeNonMatches in concatenation (.) or string at AftrRAD.pl line 958.
The barcodes you entered for data file 1 (file AllSamples.txt) are...
 TATCCG TGTATG TACATT ACTTCC AGATTT GATGTT ATTGAC CGCCCT CTGAAC TAGACA TTTACA TTCTTT GATTCG TTCACG AAAGAC GCCAGA TAACAC GCTATG GGGTAT GGTCAC CGATGG GGCACT AACCAT AGAAGC 

The names you entered for data file 1 (file AllSamples.txt) are...
 1201.fq 1202.fq 1203.fq 1204.fq 1205.fq 2485.fq 2486.fq 2487.fq 2488.fq 2489.fq 5033.fq 5034.fq 5035.fq 5036.fq 5037.fq 6098.fq 6099.fq 6100.fq 6101.fq 6102.fq 6233.fq 6234.fq 6235.fq 6236.fq 

Filtering sequence 1000000.
Filtering sequence 2000000.
Filtering sequence 3000000.
Filtering sequence 4000000.
Filtering sequence 5000000.
Filtering sequence 6000000.
Filtering sequence 7000000.
Filtering sequence 8000000.
Filtering sequence 9000000.
Filtering sequence 10000000.
Filtering sequence 11000000.
Filtering sequence 12000000.
Filtering sequence 13000000.
Filtering sequence 14000000.
Filtering sequence 15000000.
Filtering sequence 16000000.
Filtering sequence 17000000.
Filtering sequence 18000000.
Filtering sequence 19000000.

Demultiplexing samples for data file AllSamples.txt.

Identifying unique sequences for each individual.

Identifying all unique sequences within the dataset.

No such file or directory at AftrRAD.pl line 1408.
Creating file to test mean read counts.

Thanks,
Thomas

​

[Mike Sovic]

Hi Thomas,

1.)  You were right in removing the 'DataPath' argument - the default should be used here when using the dplexedData flag.  I can see where this can be confusing in the manual, so I'll get it updated in the next version.

2.)  The error "Use of uninitialized value $TotalBarcodeNonMatches…" at lines 957 and 958 shouldn't be a problem - it's a bug associated with running demultiplexed data, and again, I'll get it fixed in the next version, but I think you can safely ignore this for now. It's just dealing with printing a line in the Report file, so nothing crucial.

3.) I think the error associated with line 1408 is the one we need to deal with - we'll see if we can track it down. Check first to see if you have a set of files in the TempFiles folder named "UniqueWithCountsIndividualX.txt", where X represents each of your sample names, and that these files contain data.  If these don't exist, next check to see that you have a file named "AllUniquesSorted.txt" (also in the TempFiles folder), and again, that it contains data (sequences).  Let us know about these, and then we'll go from there.

                  Mike