Barcodes with different length size.

[jose luis acosta]

Dear Mike Sovic ,

In my barcode file, I have different sequence sizes, for example:

 Sample   Barcode

ALT01 AACCGAACT

ALT02 AGAGTAGGAT

ALT03 CAATCTAATA

ALT04 CGTCGCGCGGT

ALT05 GATCGTTCAGA

ALT06 GCTTGATTGGAT

ALT07 TATTGACGACTAC

ALT08 TGCGCGGATTGGT

ALT09 AAGACAGGC

ALT10 AGGCTCCTAC

ALT11 CAGCGTACAT

ALT12 CTCAGCTTCCA

ALT14 GCAGGAAAAGTT

ALT15 GGCGTCTACGGA

ALT16 TCATCCCATGGGT

ALT17 TGCTAGTGAGGGT

ALT18 AAGGTCGAT

ALT19 ATAAGCTGTA

ALT20 CCATATGCGA

ALT21 CTCGCGCCAGT

ALT22 GCCTTAACGCCT

ALT23 GGTCCGAACTTC

ALT24 TCCGGCCGGATAT

ALT25 TTACTTAGGCCAT

when I run ./AftrRAD.pl script, only I can put a same barcode size.

How to, I can modify  ./AftrRAD.pl script for allow different sizes of barcodes??

best wishes

Jose Luis Acosta Ph. D.

[Mike Sovic]

Hi Jose,

There should not be any problem with having different barcode sizes - this will be recognized from the barcode file automatically and accounted for.  There is more information about this in the FAQ document (attached).  Having said that, our lab has always used barcodes with the same length, so we haven't had the opportunity to test this function out a great deal ourselves.  I'm pretty sure other labs have run analyses with varying barcode lengths, and I'm not aware of any problems, at least up through version 4.1.  If you see any specific errors/warnings when running AftrRAD with your dataset that might be associated with the varying barcode lengths, or if you have any concerns about the dataset that is produced, please let us know.  Good luck!

           Mike      

[jose luis acosta]

hI Mike

I put X at the end of the different barcodes, this can work ??  example:

AACCGAACTXXXX ALT01

AGAGTAGGATXXX ALT02

CAATCTAATAXXX ALT03

CGTCGCGCGGTXX ALT04

GATCGTTCAGAXX ALT05

GCTTGATTGGATX ALT06

TATTGACGACTAC ALT07

TGCGCGGATTGGT ALT08

AAGACAGGCXXXX ALT09

AGGCTCCTACXXX ALT10

CAGCGTACATXXX ALT11

CTCAGCTTCCAXX ALT12

GCAGGAAAAGTTX ALT14

GGCGTCTACGGAX ALT15

TCATCCCATGGGT ALT16

TGCTAGTGAGGGT ALT17

AAGGTCGATXXXX ALT18

ATAAGCTGTAXXX ALT19

CCATATGCGAXXX ALT20

CTCGCGCCAGTXX ALT21

GCCTTAACGCCTX ALT22

GGTCCGAACTTCX ALT23

TCCGGCCGGATAT ALT24

TTACTTAGGCCAT ALT25

AATAGCTCCXXXX COL01

ATTCAGGAACXXX COL02

CCGACTTCTCXXX COL03

CTGCCGCTCTAXX COL04

GCGAAAATATGCX COL05

GTAATGAATTCAX COL06

TCGTACGCGGAGA COL07

TTAGCTATCGGGA COL08

ACCAGGCGTXXXX COL09

ATTGGGGTGTXXX COL10

CGATTATCGTAXX COL11

CTTAAGGTTGTXX COL12

GCTCTATGAAACX COL13

GTCTATAGCGGAX COL14

TCTACCGTGTGGT COL15

TTATGTCTCAGTC COL16

ACCTATCACXXXX COL17

CAACTGTATTXXX COL18

CGGCACCAGCTXX COL19

GATATTGGCTAXX COL20

GCTGCATTAATTX COL21

TATCTAACCGAGA COL22

TGAGAGCTGTGGA COL24

TTCGATGGTACGT COL25

best regards

jose

[Mike Sovic]

Hi Jose,

No, putting X's at the end of the barcodes will not work.  Just remove the X's from what you have here and what is left should work - again, it should be OK if the barcodes are different lengths.

               Mike

[jose luis acosta]

Hi Mike,

I run AftrRAD.pl script and obtained this error:

Scoring each pairwise alignment.  Alignments exceeding 90 % similarity will be retained.

Parsing aligned sequences into candidate loci.

Finished writing the mafft output.

Identifying and removing paralogous loci.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $SecondCount in numeric ge (>=) at AftrRAD.pl line 2719.

Use of uninitialized value $ThirdCount in numeric ge (>=) at AftrRAD.pl line 2736.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $SecondCount in numeric ge (>=) at AftrRAD.pl line 2719.

Use of uninitialized value $ThirdCount in numeric ge (>=) at AftrRAD.pl line 2736.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $SecondCount in numeric ge (>=) at AftrRAD.pl line 2719.

Use of uninitialized value $ThirdCount in numeric ge (>=) at AftrRAD.pl line 2736.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $SecondCount in numeric ge (>=) at AftrRAD.pl line 2719.

Use of uninitialized value $ThirdCount in numeric ge (>=) at AftrRAD.pl line 2736.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $SecondCount in numeric ge (>=) at AftrRAD.pl line 2719.

Use of uninitialized value $ThirdCount in numeric ge (>=) at AftrRAD.pl line 2736.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $SecondCount in numeric ge (>=) at AftrRAD.pl line 2719.

Use of uninitialized value $ThirdCount in numeric ge (>=) at AftrRAD.pl line 2736.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $SecondCount in numeric ge (>=) at AftrRAD.pl line 2719.

Use of uninitialized value $ThirdCount in numeric ge (>=) at AftrRAD.pl line 2736.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $SecondCount in numeric ge (>=) at AftrRAD.pl line 2719.

Use of uninitialized value $ThirdCount in numeric ge (>=) at AftrRAD.pl line 2736.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value in addition (+) at AftrRAD.pl line 2709.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $CurrentLocus in concatenation (.) or string at AftrRAD.pl line 2900.

Use of uninitialized value $PreviousLocusNumber in concatenation (.) or string at AftrRAD.pl line 3103, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[0] in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[1] in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value $PreviousLocusNumber in concatenation (.) or string at AftrRAD.pl line 3103, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[0] in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[1] in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value $PreviousLocusNumber in concatenation (.) or string at AftrRAD.pl line 3103, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[0] in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[1] in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value $PreviousLocusNumber in concatenation (.) or string at AftrRAD.pl line 3103, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[0] in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[1] in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value $PreviousLocusNumber in concatenation (.) or string at AftrRAD.pl line 3103, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[0] in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[1] in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value $PreviousLocusNumber in concatenation (.) or string at AftrRAD.pl line 3103, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[0] in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3104, <READFILE> line 1.

Use of uninitialized value $TempSeqArray[1] in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value in concatenation (.) or string at AftrRAD.pl line 3105, <READFILE> line 1.

Use of uninitialized value $PreviousLocusNumber in concatenation (.) or string at AftrRAD.pl line 3103, <READFILE> line 1.

I had observed this error before, for this reason, I consulted you.

any idea??

best regards

Jose

[Mike Sovic]

Hi Jose,

OK - we'll try to find the problem, but we will need some more information.  At this point in the script, you should have the folder TempFiles/RawReadCountFiles.  Inside this folder, there should be a set of files named "RawReadCounts_X.txt", where X is one of your sample names.  First, check to make sure all of these files are approximately the same size.  If so, open one of them (it doesn't matter which one), and send us the last ~10-20 lines of this file.  Once we see what those look like, we'll go from there.

          Mike

[jose luis acosta]

Hi Mike,

All files were created , but are empty. What is this about??

best regards

Jose

[Mike Sovic]

Hi Jose,

Not sure yet - next check the folder TempFiles/ErrorReadTest.  This folder should contain the files "AllReadsAndDepths.txt" and "ErrorTestOut.txt".  Check to make sure there are sequences in both of these and let me know.  If these do contain sequences, send the first ~50 lines or so from the ErrorTestOut.txt file.

              Mike 

[jose luis acosta]

Hi Mike,

AllReadsAndDepths.txt  file content is:

AAACGAAGGAGAGCTGGCGATTGCGCATCAGCGCGCCCGGCAGAAGGCCGGCTTCGAGCAGGATCTGGAAACCGTTGCACAC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AAAGCTAGGTCAATGGTGGAAACATGCACCATACTAGCTAGCCGGACACAGAGTACCTCCCCACCAAAGGGCAGAGATCGGA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

AAATGTAAATATATATAGGGAAGAGACTGTAGGGTTCATGGGAAATGTCTATTGGCAGTTGAGACTTTAAAGTGGGTAGTTT 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0

AACATCTTAGTAGCCAGAGGAAAAGAAAGCAAAAGCGATTCCCGTAGTAGCGGCGAGCGAAATGGGAGCAGAGATCGGAAGA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

AACCTTAACCGATGGTCAAGTAGACAAGTTTGAGAAGGGAGGATTAGCTACCATTGCAATAATGGTAGCACAGGCAGAGATC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0

AACGCGGTGATCTACCGTATGCGAACAGGGTGCCAGTGGAACCACCTTCCCAAAGAGTTTCCCGACGATGCCTCGGTGCACC 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AAGAAATAGACCAATCTTGTGCTCATCCTCTTGAACCGGCCTCTGTATCTGAGTCCGTGAAGGTTTTGACTGAGGGCCTGGC 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AAGCTCAGAAGGGAGAAGAGAACTCGGACAGCTCTCGTTTATAGAGACTCTAGTAAATATCATCCGGGACGGTAGTCCAAAG 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0

AATATGGGTGTCCGATCCTTATGGACTAACGGGAAAAATACAACCTGTAAATCCGGCGTGGGGCGTGGAAGGTTTTGATCCT 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AATGGCTAGTACTGCAGTCGGCCTTGCCTAAACACTTTGATCTCAGGGCCGTGCGTCGAGAAGGAGCCGAAATTGAACATTA 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AATTAACAGCAAATGGTTATGATAGCACTTGAGCTAATTTGTTGATTTAAATTGAGCCCCACGTAGAATATACAGACAAGCT 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

AATTACCTTCAGTTTAAATGACTCCAGTTGTTTCTCTGAATCTCTTGTCAGGCGTTCAACCTCCGCACATGCAATTCTCCTT 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

The ErrorTestOut.tx file is empty . This error does not occur when the barcode file was separated by length , ie , the same size of sequence. The analysis this way does not help me .

Jose

[Mike Sovic]

Hi Jose,

This AllReadsAndDepths file contains every unique sequence read in the dataset, and the values represent the number of times that sequence read occurred in each of your samples.  For each read, these values are averaged (ignoring any zeros) to determine whether the read is included in the ErrorTestOut.txt file.  If the average for the read is greater than the MinDepth parameter (default is 5 I think), then the read is printed to the ErrorTestOut.txt file, and is used for the next steps.  Otherwise, the read is discarded.  All of the reads you sent have an average of 1, so they would be discarded (presumably error reads).  

I assume what you sent is just a sample from the AllReadsAndDepths.txt file - right?  If so, can you scroll through this file and make sure there are reads that have an average of 5 or more?  I'm guessing there are if this worked previously with a subset of your barcodes, but want to make sure before we go further.

                         Mike