single reads

[Bindu Naik]

Hello,

 I'm all new to LotuS and have very less knowledge about how it works and the scripts used. I have few basic queries. The sample I have is demultiplexed single reads. I tried giving the following command which failed in demultiplexing step. It's obvious that it will fail in demultiplexing because the samples are already demultiplexed. Is there any other option to process the single read files? Looking forward for help. Thank you.

=========================================================================

          

          LotuS 1.565

=========================================================================

COMMAND

perl lotus.pl -i ./case -map mapfile_case -s /home/genetics/Desktop/lotus_pipeline/sdm_miSeq.txt -o ./case_output -c /home/genetics/Desktop/lotus_pipeline/lOTUs.cfg -threads 8 -highmem 1 -p miseq -derepMin 3:2 -simBasedTaxo lambda

Usearch ver 7 is outdated, it is recommended to install ver 9.

Download from http://drive5.com/ and execute 

"./autoInstall.pl -link_usearch [path to usearch9]"

Checking for updates..  Your LotuS version is up-to-date!

=========================================================================

          Reading mapping file

=========================================================================

Sequence files are indicated in mapping file.

Running UPARSE de novo sequence clustering..

Running fast LotuS mode..

------------ I/O configuration --------------

Input=   ./case

Output=  ./case_output

TempDir= ./case_output/tmpFiles/

------------ Configuration LotuS --------------

Sequencing platform=miseq

AmpliconType=SSU

OTU id=0.97

min unique read abundance=3:2

UCHIME_REFDB, ABSKEW=/home/genetics/Desktop/lotus_pipeline//DB//rdp_gold.fa, 2

OTU, Chimera prefix=OTU_, CHIMERA_

Similarity search with Lambda

ReferenceDatabase=greengenes

RefDB location=/home/genetics/Desktop/lotus_pipeline//DB//gg_13_5.fasta

TaxonomicGroup=bacteria

--------------------------------------------

=========================================================================

          Demultiplexing input files

           elapsed time: 2 s

=========================================================================

FAILED sdm demultiplexing step: /home/genetics/Desktop/lotus_pipeline//sdm -i_path ./case  -o_fna ./case_output/tmpFiles//demulti.fna -sample_sep ___  -log ./case_output/LotuSLogS//demulti.log -map mapfile_case -options /home/genetics/Desktop/lotus_pipeline/sdm_miSeq.txt  -o_dereplicate ./case_output/tmpFiles//derep.fas -dere_size_fmt 0 -min_derep_copies 3:2 -suppressOutput 1  -o_qual_offset 33 -paired 1  -maxReadsPerOutput 4000000 -oneLineFastaFormat 1

Regards, 

Bindu

[Falk Hildebrand]

Hey Bindu,

can you check on the lotus webpage the mapping file configuration? Here I have some examples, of how you can setup the mapping file to take as input already demultiplexed files.

http://psbweb05.psb.ugent.be/lotus/documentation.html#MapFile

e.g. add the column "fastqFile".

if this doesn't work, please post next time also the sdm error as if occurs on your console, the lotus log file doesn't contain the necessary information to see the error within 

hth,

Falk

[Bindu Naik]

Hey Falk,

I had prepared the mapping file looking into the information that you have provided in the webpage. I have attached the mapping file. Correct me if I have made any mistake in it. Thank you.

Regards,

Bindu.

[Falk Hildebrand]

Hey Bindu,

sorry for the late answer, I was travelling the last 3 weeks.

the header might be the problem:

#SampleID BarcodeSequence LinkerPrimerSequence fastqFile Age Description

Sample.1 ERR1073139.fastq 9 Control_1

Since you don't have a Barcode Seq, this might potentially cause problems. Further, the LinkerPrimerSequence might also cause problems. Just try to run lotus with a mapping file like this:

#SampleID BarcodeXXSequence LinkerPrimXXerSequence fastqFile Age Description

hth,

Falk