This makes it possible to easily analyze hiSeq amplicon data on a laptop, by any researcher. LotuS is aimed at scientists and bioinformatician that want a simple pipeline that is streamlined to a core functionality of creating OTU and taxa abundance tables, at very fast speeds e. LotuS does not include numerical analysis of samples, instead we designed LotuS output to be easily integrateable into existing workflows in e. Several quality filtering tests are included and sequences can be truncated based on accumulated error rates or quality windows falling below a threshold. LotuS workflow Advantages of LotuS Simple installation and updates of pipeline with installation script, no system variables need to be modified. One command executes pipeline.
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Filter whole sequence if one window of quality scores is below QualWindowThreshhold TrimWindowWidth, TrimWindowThreshhold Trim the end of a sequence if a window falls below quality threshhold. Set fastqVersion to 2, if you use Illumina 1.
TechnicalAdapter if one or more files have a technical adapter still included e. Note that the format will be automatically detected AmpliconShortPE This option should be "T" if your amplicons are possibly shorter than a read in a paired end sequencing run e. This works in conjunction with keepPrimerSeq should be "F" and a the ReversePrimer field in the mapping file. CheckForReversedSeqs this option should be "T" if youwant to double check, if sometimes reads are completely reverse-translated.
FAQ Q: Is there a windows version? A: No. The main problem is here that some of the properitary software used in LotuS is not Windows compatible. Therefore we will not release a Windows version any time soon. Q: I need to upload my demultiplexed data to a public repository, how do I get the demultiplexed files?
A: Use the option "-saveDemultiplex 1". Q: What is the difference between sdm and LotuS? A: sdm is an integral part of LotuS, responsible for quality filtering, demultiplexing, sequence format changes and seed extension. However, sdm was conceptualized as a stand-alone software. I personally use sdm to quality filter sequence files before assembling bacterial genomes.
To get more information of the sdm interface, execute the sdm binary without arguments ". Q: Can I use gzip compressed files? A: Yes. For config and mapping files this is not supported but all sequence files can be compressed using gzip. Just make sure the file ending is ".
Note that on some systems sdm compilation with zlib library may fail; the autoinstaller attempts to detect this and compile sdm without zlib support.
Q: What part of the sequence is cut? A: Everything that is the remainder of technical processes is removed, if possible. Giving Barcodes in the sequence, will remove all sequence upstream of the Barcodes including heterogenity spacer, illumina primer.
If Fwd and Rev 16S amplification Primers are provided in the mapping file and they are found in a read , everything upstream of these is removed including Barcodes, het spacer etc.
A: In general we do not recommend this, as these sequences could be environmental sequences that are not 16S e. If you assume that you might have new phyla in your sample or species very distant from known organisms, you can deactivate this option, but I would still recommend to cross check with e. This option is activated by default, because it was confusing if a large part of reads went silently missing. Q: My Barcodes are reverse complemented, can I set an option to take care of this?
A: This should be automatically detected: sdm has an inbuilt algorithm that checks in the first sequences, if more reverse complemented BCs can be detected and will use this information for the rest of the file. However, BCs have to be consistent in their direction, as the direction information is assummed to be the same within each file. Q: I do not want to use RDP assigned taxonomies, but use reference databases.
A: Both databases have a large selection of taxa included, though SILVA has a faster release cycle and is currently more up to date, the last GG release was in Q: How to choose a good cutoff length of sequences? A: Changing the TruncateSequenceLength and minSeqLength is fine tuning to your data set - just remember to keep these parameters equal. As a general rule of thumb: you want to have as long as possible reads, but every read below that length will be excluded.
Further, the accumulated error has to be below e. A: First of all you need to optimize the number of sequences you gain vs the number of errors you allow to pass into OTU building. Second, choose your clustering algorithm according to your needs. UPARSE is my general recommendation; some users have reported better clusterings in the usearch7x versions. SEED clusters are very sensitive, to a point where read errors could cluster into a new OTU, but if you need pseudo-strain resolution this might work for you.
These are plain "good old clusters". Third, think about what taxonomic assignments you need and from which database. Depends on the environment you work in. So for gut environment, you should get a good fraction of OTUs assigned to species level; other environments like Arctic samples are often not well represented at species level.
Further, if the identity of the hit is below a certain threshold set in the lotus. This is to prevent falsely assigned species names, even if this means retuning a lot of genera without species assignments. Q: Where are reads exactly removed during the LotuS run A: 1st during Quality filtering and also dereplication these are later counted into the OTU matrix by similarity comparison, but not used for OTU construction.
Q: How can I cite LotuS in my work? LotuS: an efficient and user-friendly OTU processing pipeline. Microbiome 2:
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Filter whole sequence if one window of quality scores is below QualWindowThreshhold TrimWindowWidth, TrimWindowThreshhold Trim the end of a sequence if a window falls below quality threshhold. Set fastqVersion to 2, if you use Illumina 1. TechnicalAdapter if one or more files have a technical adapter still included e. Note that the format will be automatically detected AmpliconShortPE This option should be "T" if your amplicons are possibly shorter than a read in a paired end sequencing run e.
Tutorial: 30 LotusScript tips
The Lotus Approach Barcode Generation Tutorial contains information, examples and steps needed for generating barcodes in Lotus Approach. The appropriate barcode fonts must be installed before the application can display or print barcodes. For environment-specific installation instructions, please refer to these font installation procedures. Simply create a field computed for display that combines the start and stop characters with the field. Enter the formula in the Modification Formula box. Choose the appropriate barcode font for the newly created field, making sure that it is centered in the box with some white space before and after the barcode to ensure a clean scan. A separate field must be created that will contain the barcode.
LotusScript Classes Tutorial