Difference between revisions of "Fastq mcf"

Revision as of 17:31, 25 March 2019

Introduction

fastq-mcf attempts to:

• Detect & remove sequencing adapters and primers
• Detect limited skewing at the ends of reads and clip
• Detect poor quality at the ends of reads and clip
• Detect Ns, and remove from ends
• Remove reads with CASAVA 'Y' flag (purity filtering)
• Discard sequences that are too short after all of the above
• Keep multiple mate-reads in sync while doing all of the above

Usage

Usage: fastq-mcf [options] <adapters.fa> <reads.fq> [mates1.fq ...]
Detects levels of adapter presence, computes likelihoods and locations (start, end) of the adapters. Removes the adapter sequences from the fastq file(s).

Stats go to stderr, unless -o is specified.

Specify -0 to turn off all default settings

If you specify multiple 'paired-end' inputs, then a -o option is required for each. IE: -o read1.clip.q -o read2.clip.fq

Options:

   -h       This help
   -o FIL   Output file (stats to stdout)
   -s N.N   Log scale for adapter minimum-length-match (2.2)
   -t N     % occurance threshold before adapter clipping (0.25)
   -m N     Minimum clip length, overrides scaled auto (1)
   -p N     Maximum adapter difference percentage (10)
   -l N     Minimum remaining sequence length (19)
   -L N     Maximum remaining sequence length (none)
   -D N     Remove duplicate reads : Read_1 has an identical N bases (0)
   -k N     sKew percentage-less-than causing cycle removal (2)
   -x N     'N' (Bad read) percentage causing cycle removal (20)
   -q N     quality threshold causing base removal (10)
   -w N     window-size for quality trimming (1)
   -H       remove >95% homopolymer reads (no)
   -0       Set all default parameters to zero/do nothing
   -U|u     Force disable/enable Illumina PF filtering (auto)
   -P N     Phred-scale (auto)
   -R       Dont remove Ns from the fronts/ends of reads
   -n       Dont clip, just output what would be done
   -C N     Number of reads to use for subsampling (300k)
   -S       Save all discarded reads to '.skip' files
   -d       Output lots of random debugging stuff

Quality adjustment options:

   --cycle-adjust    CYC,AMT     Adjust cycle CYC (negative = offset from end) by amount AMT
   --phred-adjust    SCORE,AMT   Adjust score SCORE by amount AMT

Filtering options:

   --[mate-]qual-mean  NUM       Minimum mean quality score
   --[mate-]qual-gt    NUM,THR   At least NUM quals > THR
   --[mate-]max-ns     NUM       Maxmium N-calls in a read (can be a %)
   --[mate-]min-len    NUM       Minimum remaining length (same as -l)
   --hompolymer-pct    PCT       Homopolymer filter percent (95)

If mate- prefix is used, then applies to second non-barcode read only

Adapter files are 'fasta' formatted:

Specify n/a to turn off adapter clipping, and just use filters

Increasing the scale makes recognition-lengths longer, a scale of 100 will force full-length recognition of adapters.

Adapter sequences with _5p in their label will match 'end's, and sequences with _3p in their label will match 'start's, otherwise the 'end' is auto-determined.

Skew is when one cycle is poor, 'skewed' toward a particular base. If any nucleotide is less than the skew percentage, then the whole cycle is removed. Disable for methyl-seq, etc.

Set the skew (-k) or N-pct (-x) to 0 to turn it off (should be done for miRNA, amplicon and other low-complexity situations!)

Duplicate read filtering is appropriate for assembly tasks, and never when read length < expected coverage. -D 50 will use 4.5GB RAM on 100m DNA reads - be careful. Great for RNA assembly.

Quality filters are evaluated after clipping/trimming