What it does
Most modern sequencing technologies produce reads that have deteriorating quality towards the 3'-end and some towards the 5'-end as well. Incorrectly called bases in both regions negatively impact assembles, mapping, and downstream bioinformatics analyses.
Sickle is a tool that uses sliding windows along with quality and length thresholds to determine when quality is sufficiently low to trim the 3'-end of reads and also determines when the quality is sufficiently high enough to trim the 5'-end of reads. It will also discard reads based upon the length threshold. It takes the quality values and slides a window across them whose length is 0.1 times the length of the read. If this length is less than 1, then the window is set to be equal to the length of the read. Otherwise, the window slides along the quality values until the average quality in the window rises above the threshold, at which point the algorithm determines where within the window the rise occurs and cuts the read and quality there for the 5'-end cut. Then when the average quality in the window drops below the threshold, the algorithm determines where in the window the drop occurs and cuts both the read and quality strings there for the 3'-end cut. However, if the length of the remaining sequence is less than the minimum length threshold, then the read is discarded entirely (or replaced with an "N" record). 5'-end trimming can be disabled. Sickle also has an option to truncate reads with Ns at the first N position.
Sickle supports three types of quality values: Illumina, Solexa, and Sanger. Note that the Solexa quality setting is an approximation (the actual conversion is a non-linear transformation). The end approximation is close. Illumina quality refers to qualities encoded with the CASAVA pipeline between versions 1.3 and 1.7. Illumina quality using CASAVA >= 1.8 is Sanger encoded. The quality value will be determined from the datatype of the data, i.e. a fastqsanger datatype is assumed to be Sanger encoded.
Note that Sickle will remove the 2nd FASTQ record header (on the "+" line) and replace it with simply a "+". This is the default format for CASAVA >= 1.8.
Options
Single-end
This option takes one single-end input file and outputs one single-end output file of reads that passed the filters.
Paired-End (one interleaved input file)
This option takes as input one interleaved paired-end file. If you then check the "Output only one file with all reads" checkbox, it will output one interleaved file where any read that did not pass filter will be replaced with a FASTQ record where the sequence is a single "N" and the quality is the lowest quality possible for that quality type. This will preserve the paired nature of the data. If you leave the checkbox unchecked, it will output two files, one interleaved file with all the passed pairs and one singletons file where only one of the pair passed filter.
Paired-End (two separate input files)
This option takes two separate (forward and reverse) paired-end files as input. The output is three files: Two paired-end files with pairs that passed filter and a singletons file where only one of the pair passed filter.
Quality threshold
Input your desired quality threshold. This threshold is phred-scaled, which is typically values between 0-41 for FASTQ data.
Length threshold
Input your desired length threshold. This is the threshold to determine if a read is kept after all the trimming steps are done.
Disable 5-prime trimming
An option to disable trimming the read on the 5-prime end. This trimming trims the read if the average quality values dip below the quality threshold at the 5-prime end.
Truncate sequences with Ns
This option will trim a read at the first "N" base in the read after doing quality trimming. It is then still subject to the length threshold.
Copyright: Nikhil Joshi