The workflow sample, described below, takes FASTQ files with metagenomic NGS reads as input and process them as follows:

• Improve reads quality with Trimmomatic
• Provide FastQC reads quality reports
• Classification:
  1. Classify the pre-processed reads with Kraken
  2. Classify the assembled contigs with CLARK
  3. Classify these reads with DIAMOND (in case of SE reads)
  4. Classify these reads with MetaPhlAn2
  5. Ensemble the classification output from Kraken, CLARK, and DIAMOND into a CSV file.
  6. Improve classification from these tools with WEVOTE.
  7. Provide general classification reports

Workflow Sample Location

The workflow sample "Parallel NGS Reads Classification" can be found in the "NGS" section of the Workflow Designer samples.

Workflow Image

The opened workflow for single-end reads looks as follows:

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  <img src="/wiki/download/attachments/22061357/Parallel NGS Reads Classification_0.jpg"/>
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The opened workflow for paired-end reads looks as follows:

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  <img src="/wiki/download/attachments/22061357/Parallel NGS Reads Classification_1.jpg"/>
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Workflow Wizard

The wizard has 6 pages.

1. Input data: On this page, input files must be set. 

   <center> <br> <img src="/wiki/download/attachments/22061357/Parallel NGS Reads Classification_2.jpg"/> <br> </center>

2. Trimmomatic settings: The Trimmomatic parameters can be changed here.

   <center> <br> <img src="/wiki/download/attachments/22061357/Parallel NGS Reads Classification_3.jpg"/> <br> </center>

   To configure trimming steps use the following button:

   <center> <br> <img src="/wiki/download/attachments/22062035/De novo Assemble Illumina PE Reads_3.jpg"/> <br> </center>

   The following dialog will appear:

   <center> <br> <img src="/wiki/download/attachments/22059547/Improve Reads with Trimmomatic Element.png"/> <br> </center>

   Click the Add new step button and select a step. The following options are available:

   • ILLUMINACLIP: Cutadapterandotherillumina-specific sequences from the read.
   • SLIDINGWINDOW: Perform a sliding window trimming, cutting once the average quality within the window falls below a threshold.
   • LEADING: Cut bases off the start of a read, if below a threshold quality.
   • TRAILING: Cut bases off the end of a read, if below a threshold quality.
   • CROP: Cut the read to a specified length.
   • HEADCROP: Cut the specified number of bases from the start of the read.
   • MINLEN: Drop the read if it is below a specified length.
   • AVGQUAL: Drop the read if the average quality is below the specified level.
   • TOPHRED33: Convert quality scores to Phred-33.
   • TOPHRED64: Convert quality scores to Phred-64.

   Each step has the own parameters:

   AVGQUAL

   This step drops a read if the average quality is below the specified level.

   Input the following values:

   • Quality threshold: the minimum average quality required to keep a read.

   CROP

   This step removes bases regardless of quality from the end of thread, so that the readhas maximally the specified length after this step has been performed. Steps performed after CROP might of course further shorten the read.

   Input the following values:

   • Length: the number of bases to keep, from the start of the read.

   HEADCROP

   This step removes the specified number of bases, regardless of quality, from the beginning of the read.

   Input the following values:

   • Length: the number of bases to remove from the start of the read.

   ILLUMINACLIP

   This step is used to find and remove Illumina adapters.

   Trimmomatic first compares short sections of an adapter and a read. If they match enough, the entire alignment between the read and adapter is scored. For paired-end reads, the "palindrome" approach is also used to improve the result. See Trimmomatic manual for details.

   Input the following values:

   • Adapter sequences: a FASTA file with the adapter sequences. Files for TruSeq2 (GAII machines), TruSeq3 (HiSeq and MiSeq machines) and Nextera kits for SE and PE reads are now available by default. The naming of the various sequences within the specified file determines how they are used.
   • Seed mismatches: the maximum mismatch count in short sections which will still allow a full match to be performed.
   • Simple clip threshold: a threshold for simple alignment mode. Values between 7 and 15 are recommended. A perfect match of a 12 base sequence will score just over 7, while 25 bases are needed to score 15.
   • Palindrome clip threshold: a threshold for palindrome alignment mode. For palindromic matches, a longer alignment is possible. Therefore the threshold can be in the range of 30. Even though this threshold is very high (requiring a match of almost 50 bases) Trimmomatic is still able to identify very, very short adapter fragments.

   There are also two optional parameters for palindrome mode: Min adapter length and Keep both reads. Use the following dialog. To call the dialog press the Optional button.

   <center> <br> <img src="/wiki/download/attachments/22059547/Improve Reads with Trimmomatic Element_1.jpg"/> <br> </center>

   LEADING

   This step removes low-quality bases from the beginning. As long as a base has a value below this threshold the base is removed and the next base will be investigated.

   Input the following values:

   • Quality threshold: the minimum quality required to keep a base.

   MAXINFO

   This step performs an adaptive quality trim, balancing the benefits of retaining longer reads against the costs of retaining bases with errors. See Trimmomatic manual for details.

   Input the following values:

   • Target length: the read length which is likely to allow the location of the read within the target sequence. Extremely short reads, which can be placed into many different locations, provide little value. Typically, the length would be in the order of 40 bases, however, the value also depends on the size and complexity of the target sequence.
   • Strictness: the balance between preserving as much read length as possible vs. removal of incorrect bases. A low value of this parameter (0.8) favours read correctness.

   MINLEN

   This step removes reads that fall below the specified minimum length. If required, it should normally be after all other processing steps. Reads removed by this step will be counted and included in the "dropped reads" count.

   Input the following values:

   • Length: the minimum length of reads to be kept.

   SLIDINGWINDOW

   This step performs a sliding window trimming, cutting once the average quality within the window falls below a threshold. By considering multiple bases, a single poor quality base will not cause the removal of high-quality data later in the read.

   Input the following values:

   • Window size: the number of bases to an average across.
   • Quality threshold: the average quality required.

   TOPHRED33

   This step (re)encodes the quality part of the FASTQ file to base 33.

   TOPHRED64

   This step (re)encodes the quality part of the FASTQ file to base 64. 

   TRAILING

   This step removes low-quality bases from the end. As long as a base has a value below this threshold the base is removed and the next base (i.e. the preceding one) will be investigated. This approach can be used removing the special Illumina " low-quality segment" regions (which are marked with a quality score of 2), but SLIDINGWINDOW or MAXINFO are recommended instead.

   Input the following values:

   • Quality threshold: the minimum quality required to keep a base.

   To remove a step use the Remove selected step button. The pink highlighting means the required parameter has not been set.

    

3. Kraken settings: Default Kraken parameters can be changed here. 

   <center> <br> <img src="/wiki/download/attachments/22061357/Parallel NGS Reads Classification_4.jpg"/> <br> </center>

   The following parameters are available:

   Database	A path to the folder with the Kraken database files.
   Quick operation	Stop classification of an input read after the certain number of hits. The value can be specified in the "Minimum number of hits" parameter.

4. CLARK settings: Default CLARK parameters can be changed here. 

   <center> <br> <img src="/wiki/download/attachments/22061357/Parallel NGS Reads Classification_5.jpg"/> <br> </center>

   The following parameters are available:

   Database	A folder that should be used to store the database files.
   K-mer length	This value is critical for the classification accuracy and speed. For high sensitivity, it is recommended to set this value to 20 or 21 (along with the "Full" mode). However, if the precision and the speed are the main concern, use any value between 26 and 32. Note that the higher the value, the higher is the RAM usage. So, as a good tradeoff between speed, precision, and RAM usage, it is recommended to set this value to 31 (along with the "Default" or "Express" mode).
   Minimumk-merfrequency	Minimum of k-mer frequency/occurrence for the discriminative k-mers (-t). For example, for 1 (or, 2), the program will discard any discriminative k-mer that appear only once (or, less than twice).
   Mode	Set the mode of the execution (-m): "Full" to get detailed results, confidence scores, and other statistics. "Default" to get results summary and perform the best trade-off between classification speed, accuracy and RAM usage. "Express" to get results summary with the highest speed possible.
   Sampling factor value
   Gap	"Gap" or number of non-overlapping k-mers to pass when creating the database (-п). Increase the value if it is required to reduce the RAM usage. Note that this will degrade the sensitivity.

5. WEVOTE settings: DefaultWEVOTE parameters can be changed here. 

   <center> <br> <img src="/wiki/download/attachments/22061357/Parallel NGS Reads Classification_6.jpg"/> <br> </center>

   The following parameters are available:

   Penalty	Score penalty for disagreements (-k)
   Numberof agreed tools	Specify the minimum number of tools agreed on WEVOTE decision (-a).
   Score threshold	Score threshold (-s)

6. Output Files Page: On this page, you can select an output directory:

   <center> <br> <img src="/wiki/download/attachments/22061357/Parallel NGS Reads Classification_7.jpg"/> <br> </center>