Invalid barcode error
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Gemma
Aug 31, 2015, 11:35:08 AM8/31/15
to Stacks
Dear all,
I am a new user to stacks and it appears that I am falling down at the first hurdle! I am running stacks version 1.29 and trying to use process_radtags to clean and demultiplex my paired-end, single-digest RAD-seq data. It has been sequenced on an Illumina HiSeq.
I have two problems:
1. When I supply a path to the directory where my paired-end files are kept, it is unable to locate any input files so perhaps there is a problem with the names of my files? I have not changed the names so I assume they have not been altered since coming off the machine, but perhaps they have an atypical naming scheme. I would really like to be able to give a path as I have multiple directories with multiple paired files within each.
2. My barcode file is not being read properly, or my barcodes aren't being recognised.
This is what I'm using to run process_radtags and the errors I get:
gemma@zoo-stack01:~$ process_radtags -P -p ./EPKP/raw_reads/2014072_THLib1/ \
> -o ./EPKP/clean/test2/ \
> -b ./EPKP/raw_reads/2014072_THLib1/barcodes1 \
> -e sbfI -r -c -q -t 114 -i gzfastq \
> --inline_index
Using Phred+33 encoding for quality scores.
Reads will be truncated to 114bp
Unable to locate any input files to process within './EPKP/raw_reads/2014072_THLib1/'
Found 0 paired input file(s).
Searching for single-end, inlined and paired-end, indexed barcodes.
Invalid barcode on line 1: 'EPWIS012'
Here is the structure of my barcodes file (there are tabs between columns):
gemma@zoo-stack01:~$ cat EPKP/raw_reads/2014072_THLib1/barcodes1
TGTGACTG EPWIS012
ACACGACA EPWIS013
GTCATGTG EPWIS010
TGCATCGT EPWIS011
CAGTCTCA EPWIS008
GTACTCGT EPWIS009
ACGTAGCA EPWIS002
CACACAGT EPWIS007
ACGTCTAC EPWIS014
CAGTGTGT EPWIS015
CGATACTA EPGOU034
GTACATCA EPGOU035
CATGATCA EPWIS021
GTACGCTG EPWIS025
GTCAGTGT EPWIS024
TGTGTGAC EPWIS026
TGTGCAGT EPGOU030
ACGTGCTG EPGOU031
ACTGATAC EPGOU032
CATGCGAC EPGOU033
ACACTGAC EPWIS001
TATATGCG EPGOU037
GTGTACTG EPGOU036
and this is how my files are named:
gemma@zoo-stack01:~$ ls EPKP/raw_reads/2014072_THLib1
140611_D00261_0140_AC465GANXX_1_SA-PE-001_1.sanfastq.gz.md5
140611_D00261_0140_AC465GANXX_1_SA-PE-001_1.sanfastq.gz
140611_D00261_0140_AC465GANXX_1_SA-PE-001_2.sanfastq.gz.md5
140611_D00261_0140_AC465GANXX_1_SA-PE-001_2.sanfastq.gz
140611_D00261_0140_AC465GANXX_2_SA-PE-001_1.sanfastq.gz.md5
140611_D00261_0140_AC465GANXX_2_SA-PE-001_1.sanfastq.gz
140611_D00261_0140_AC465GANXX_2_SA-PE-001_2.sanfastq.gz.md5
140611_D00261_0140_AC465GANXX_2_SA-PE-001_2.sanfastq.gz
140611_D00261_0140_AC465GANXX_3_SA-PE-001_1.sanfastq.gz.md5
140611_D00261_0140_AC465GANXX_3_SA-PE-001_1.sanfastq.gz
140611_D00261_0140_AC465GANXX_3_SA-PE-001_2.sanfastq.gz.md5
140611_D00261_0140_AC465GANXX_3_SA-PE-001_2.sanfastq.gz
And in case it is needed, this is how the individuals have been barcoded:
- each individual is barcoded with an inline barcode, which is part of read 1
- each library is barcoded by an indexing barcode, which is read as a separate read (there are multiple individuals per library as you know)
- in this scheme, each pair of reads has effectively two barcodes, one that maps back to the individual and one that maps to the library
- the library barcode is demultiplexed by the Illumina pipeline as part of the conversion to FASTQ, this is what you see in the header
- the individual barcode is contained in read 1 and is demultiplexed by STACKS
- read 2 doesn't contain a barcode
I would really appreciate some help as I haven't been able to solve this by looking at previous posts. Sorry if I have forgotten to add any information.
With best wishes,
Gemma
Julian Catchen
Aug 31, 2015, 12:26:05 PM8/31/15
to stacks...@googlegroups.com, gvcl...@gmail.com
Hi Gemma,
There are two problems I can see with your approach. First, technically
you are feeding Stacks single-end, single barcode data. Although it was
originally dual barcoded, since the Illumina software did the first
demultiplexing for you, the index barcodes are no longer relevant to
Stacks, so you should specify the barcode type as --inline_null (or just
omit it and it will default to that). Instead, you specified a dual
barcode and Stacks interpreted your sample names as the second barcode.
Second, when scanning a directory for files, process_radtags will look
for files with a .fq, .fq.gz, .fastq, or .fastq.gz suffix. So you should
rename your files to use one of the common file suffixes. Or, you can
run process_radtags one file at a time using the -f option.
Best,
julian
There are two problems I can see with your approach. First, technically
you are feeding Stacks single-end, single barcode data. Although it was
originally dual barcoded, since the Illumina software did the first
demultiplexing for you, the index barcodes are no longer relevant to
Stacks, so you should specify the barcode type as --inline_null (or just
omit it and it will default to that). Instead, you specified a dual
barcode and Stacks interpreted your sample names as the second barcode.
Second, when scanning a directory for files, process_radtags will look
for files with a .fq, .fq.gz, .fastq, or .fastq.gz suffix. So you should
rename your files to use one of the common file suffixes. Or, you can
run process_radtags one file at a time using the -f option.
Best,
julian
Gemma
Sep 14, 2015, 11:10:06 AM9/14/15
to Stacks, gvcl...@gmail.com, jcat...@illinois.edu
Hi Julien,
Just to say that using inline_null sorted the problem.
I couldn't get it to work when I supplied the path to the directory, even when I renamed all the files to .fastq.gz or .fq.gz but I think that is because they had been renamed by the sequencing facility and didn't match the standard Illumina system.
Best,
Gemma
Michelle Taylor
Jan 11, 2018, 10:36:51 AM1/11/18
to Stacks
Dear Julien and team,
I'm using Stacks2 with pair end data.
We have RAD-seq data with an inline barcode on the forward and no barcode on the reverse
(we have asked the sequencing service how we are meant to identify the matching reverse reads and we await an answer).
I think that the appropriate barcode option is --inline_null (as per process_radtags manual -
"If your data are single-end or paired-end, with an inline barcode present only on the single-end")
however outputs from this are "killed" after 86M
process_radtags \
-p ./Loph_pl2_raw -P \
-o ./Loph_pl2_multipl \
-b /../../../home/zool-polychaete-popgen/zool0971/Lophelia_raw/Plate2_barcodes_edited -e pstI \
-E phred33 \
-r \
-c \
-D \
-q \
-i gzfastq --retain_header --inline_null
Processing paired-end data.
Using Phred+33 encoding for quality scores.
Found 1 paired input file(s).
Searching for single-end, inlined barcodes.
Loaded 96 barcodes (10bp).
Will attempt to recover barcodes with at most 1 mismatches.
Processing file 1 of 1 [Loph_Plate2_R1_001.fastq.gz]
Reading data from:
./Loph_pl2_raw/Loph_Plate2_R1_001.fastq.gz and
./Loph_pl2_raw/Loph_Plate2_R2_001.fastq.gz
Processing RAD-Tags...1M...2M...3M...4M...5M...6M...7M...8M...9M...10M...11M...12M...13M...14M...15M...16M...17M...18M...19M...20M...21M...22M...23M...24M...25M...26M...27M...28M...29M...30M...31M...32M...33M...34M...35M...36M...37M...38M...39M...40M...41M...42M...43M...44M...45M...46M...47M...48M...49M...50M...51M...52M...53M...54M...55M...56M...57M...58M...59M...60M...61M...62M...63M...64M...65M...66M...67M...68M...69M...70M...71M...72M...73M...74M...75M...76M...77M...78M...79M...80M...81M...82M...83M
...84M...85M...86M...Killed
Do you have any advice on where we are going wrong here?
The outputs from the above have many many discards and a few reads in the reverse folders, but few in the forward folders. See below
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Nicolas Rochette
Jan 12, 2018, 8:54:58 AM1/12/18
to Michelle Taylor, stacks...@googlegroups.com
Hi Michelle,
You are correct about the barcode option (--inline_null). Paired reads have the same name, except for the ending which is /1 or /2. You should either have received two FASTQ files (in *_R1_* and *_R2_*) or one FASTQ file with pairs of reads with the same name.
"Killed" is not an output produced by Stacks, but instead by the
operating system where you're running it, probably because it ran
out of time or of memory (or if you did a manual action that could
have killed it). I would help if you could post the time and
memory usage and how much is supposed to be available.
Best,
Nicolas
--
Stacks website: http://catchenlab.life.illinois.edu/stacks/
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Michelle Taylor
Feb 18, 2018, 7:45:07 AM2/18/18
to Stacks
Dear Nicolas,
I just wanted to post to say that you were right. It was a server time out issue. Thank you for your time and response.
I did eventually get it all up and running and I now have the unusual situation of having one plate of RAD-seq with 0.2% retained reads and the other with 87.3% retained reads... that is quite some disparity. They have been run with identical parameters so I'm mulling over what could be so different about the two plates... any ideas?
All the best, Michelle
PLATE 1
Processing paired-end data.
Using Phred+33 encoding for quality scores.
Found 1 paired input file(s).
Searching for single-end, inlined barcodes.
Loaded 96 barcodes (10bp).
Will attempt to recover barcodes with at most 1 mismatches.
Processing file 1 of 1 [Loph_Plate1_R1_001.fastq.gz]
Reading data from:
./Loph_pl1_raw/Loph_Plate1_R1_001.fastq.gz and
./Loph_pl1_raw/Loph_Plate1_R2_001.fastq.gz
Processing RAD-Tags...1M...2M...3M...4M...5M...6M...7M...8M...9M...10M...11M...12M...13M...14M...15M...16M...17M...18M..$
725901406 total reads; -79332272 ambiguous barcodes; -9849657 ambiguous RAD-Tags; +18291610 recovered; -2946651 low qual$
Closing files, flushing buffers...
Outputing details to log: './Loph_pl1_multipl/process_radtags.Loph_pl1_raw.log'
725901406 total sequences
79332272 ambiguous barcode drops (10.9%)
2946651 low quality read drops (0.4%)
9849657 ambiguous RAD-Tag drops (1.4%)
633772826 retained reads (87.3%)
PLATE 2
Processing paired-end data.
Using Phred+33 encoding for quality scores.
Found 1 paired input file(s).
Searching for single-end, inlined barcodes.
Loaded 96 barcodes (10bp).
Will attempt to recover barcodes with at most 1 mismatches.
Processing file 1 of 1 [Loph_Plate2_R1_001.fastq.gz]
Reading data from:
./Loph_pl2_raw/Loph_Plate2_R1_001.fastq.gz and
./Loph_pl2_raw/Loph_Plate2_R2_001.fastq.gz
Processing RAD-Tags...1M...2M...3M...4M...5M...6M...7M...8M...9M...10M...11M...12M...13M...14M...15M...16M...17M...18M..$
746570352 total reads; -744410356 ambiguous barcodes; -896041 ambiguous RAD-Tags; +1934907 recovered; -13764 low quality$
Closing files, flushing buffers...
Outputing details to log: './Loph_pl2_multipl/process_radtags.Loph_pl2_raw.log'
746570352 total sequences
744410356 ambiguous barcode drops (99.7%)
13764 low quality read drops (0.0%)
896041 ambiguous RAD-Tag drops (0.1%)
1250191 retained reads (0.2%)
...
Julian Catchen
Feb 19, 2018, 1:03:06 PM2/19/18
to stacks...@googlegroups.com, michell...@essex.ac.uk
Hi Michelle,
It appears your second plate does not have intact barcodes. Either you
specified them wrong, there was something wrong with the plate of
adaptors you used at the bench, or there is a systematic sequencing
error in the first 10bp of your reads. I would suggest extracting out
the first 10bp from your second plate of reads and trying to understand
if there are barcodes, or a bunch of Ns.
julian
It appears your second plate does not have intact barcodes. Either you
specified them wrong, there was something wrong with the plate of
adaptors you used at the bench, or there is a systematic sequencing
error in the first 10bp of your reads. I would suggest extracting out
the first 10bp from your second plate of reads and trying to understand
if there are barcodes, or a bunch of Ns.
julian
Scott Brainard
Feb 4, 2020, 2:46:21 PM2/4/20
to Stacks
Hi all,
I'm using Stacks the for the first time, but am also getting an invalid barcode error.
My call to process_radtags is:
(base) [igoldman@brc2 GWAS_panel]$ $STACKS/process_radtags -P -p /home/BIOTECH/igoldman/GWAS_panel/Box-1/ -o /home/BIOTECH/igoldman/GWAS_panel/demultiplexed/Box-1 -b /home/BIOTECH/igoldman/GWAS_panel/barcodes/box1.txt -e apeKI -r --inline_null
Output:
Using Phred+33 encoding for quality scores.
Found 1 paired input file(s).
Searching for single-end, inlined barcodes.
Invalid barcode: 'CTCC'
An example forward read:
@A00589:100:HLKHHDMXX:1:1101:2076:1016 1:N:0:GACTAGGAGC+TAGTACAGGA
CAGCTCATTAGTTCTGTAAATAGCCAATCTTCTGCAGGTACTTGGCTTTTTTGGTAAATCGATATTTTGCATTTTAGTATATGTGATGTATTCTTGTGCACTGGGTACGACCTTTTTTTTAAGTTGTTAATAACGTTGTAATTTAT
+
FFFF:FFFFFFFFFFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF,::F:FF:FFFFFFFFFF,,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:F
An example reverse read:
@A00589:100:HLKHHDMXX:1:1101:2076:1016 2:N:0:GACTAGGAGC+TAGTACAGGA
CAGCCAGAGGTGTGACTGTTGTGATCCAGTCTTCCAGATATGCTGGTTTTCCAAGTTTCCCTCTCAATCCGTTCACTAAATCACATAAATTACAACGTTATTAACAACTTAAAAAAAAGGTCGTACCCAGTGCACAAGAATACATCACATA
+
FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFF:FFFFFFFFFFF:FF
So these reads have an index, but they've already been demultiplexed by that identifier.
My barcode file:
(base) [igoldman@brc2 barcodes]$ cat box1.txt
CTCC Vilmorin-I04428
TTCTC Ames-7701
GCTTA Ames-7702
AACGCCT Ames-7705
AGGC Ames-7706
TCGTT Ames-7711
TGGCTA Ames-7713
TGCTGGA Ames-7715
TGCA Ames-25076
AGCCC Ames-27398
CTTCCA Ames-27399
AATATGC Ames-27404
GATC Ames-27414
ACCTAA Ames-29082
ACGTGTT Ames-29182
AACCGAGA NSL-6162
ACTA NSL-6166
GTATT NSL-6168
GAGATA NSL-6172
ACGACTAC NSL-6193
TCAC NSL-6619
ATATGT NSL-9333
ATTAATT NSL-22994
ACAGGGAA NSL-26501
CAGA NSL-26502
CTGTA NSL-26503
ATGCCT NSL-34344
GGTGT NSL-34346
AGGAT NSL-52533
ATCGTA NSL-54098
ATTGGAT NSL-65838
ACGTGGTA NSL-199856
AACT NSL-199857
ACCGT NSL-199859
TATTTTT NSL-199860
TAGCATGC NSL-199861
ATTGA NSL-199865
CATCGT NSL-199868
CATAAGT PI-163234
CCATGGGT PI-163239
GCGT PI-163240
GTAA PI-163241
CTTGCTT PI-164136
AGTGGA PI-164344
CATCT PI-164388
CGCGGT PI-164461
CGCTGAT PI-164798
CGCGGAGA PI-164942
TGCGA PI-164943
GGTTGT PI-165484
ATGAAAC PI-165522
TAGGCCAT PI-167055
CCTAC PI-167082
CTATTA PI-167143
CGGTAGA PI-167211
CGTGTGGT PI-169482
CGAT PI-169484
CCAGCT PI-169485
AAAAGTT PI-169486
TGCAAGGA PI-169487
GAGGA PI-169490
GCCAGT PI-171641
CTACGGA PI-171642
GCTGTGGA PI-171643
CGCTT PI-171644
TTCAGA PI-171645
GAATTCA PI-172890
TGGTACGT PI-172891
GGAAC PI-172893
GGAAGA PI-172894
GCGGAAT PI-173687
GGATTGGT PI-173688
TCACC PI-174202
TAGGAA PI-174205
GAACTTC PI-174206
TCTCAGTC PI-174207
GTCAA PI-174208
GTACTT PI-174828
TAGCGGA PI-175715
GTGAGGGT PI-175716
CTAGC PI-175717
GCTCTA PI-175718
GGACCTA PI-175719
CCGGATAT PI-176556
TAATA PI-176557
GTTGAA PI-176558
TCGAAGA PI-176559
TATCGGGA PI-176560
ACAAA PI-176561
CCACAA PI-176563
GTCGATT PI-176564
CGCCTTAT PI-176565
TACAT PI-176969
TAACGA PI-176970
TCTGTGA PI-177379
TTCCTGGA PI-177383
Any advice appreciated!
Thanks,
Scott
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