Colorspace reads

Cutadapt was designed to work with colorspace reads from the ABi SOLiD sequencer. Colorspace trimming is activated by the --colorspace option (or use -c for short). The input reads can be given either:

• in a FASTA file (typically extensions .csfasta or .csfa)

• in a FASTQ file

• in a .csfasta and a .qual file (this is the native SOLiD format). That is, cutadapt expects two file names in this case.

In all cases, the colors must be represented by the characters 0, 1, 2, 3. Here is an example input file in .fastq format that is accepted:

@1_13_85_F3
T110020300.0113010210002110102330021
+
7&9<&77)& <7))%4'657-1+9;9,.<8);.;8
@1_13_573_F3
T312311200.3021301101113203302010003
+
6)3%)&&&& .1&(6:<'67..*,:75)'77&&&5

Further example input files can be found in the cutadapt distribution at tests/data/solid.*. The .csfasta/.qual file format is automatically assumed if two input files are given to cutadapt, and when no paired-end trimming options are used.

Cutadapt always converts input data given as a pair of FASTA/QUAL files to FASTQ.

In colorspace mode, the adapter sequences given to the -a, -b and -g options can be given both as colors or as nucleotides. If given as nucleotides, they will automatically be converted to colorspace. For example, to trim an adapter from solid.csfasta and solid.qual, use this command-line:

cutadapt -c -a CGCCTTGGCCGTACAGCAG solid.csfasta solid.qual > output.fastq

In case you know the colorspace adapter sequence, you can also write 330201030313112312 instead of CGCCTTGGCCGTACAGCAG, and the result is the same.

Ambiguity in colorspace

The ambiguity of colorspace encoding leads to some effects to be aware of when trimming 3' adapters from colorspace reads. For example, when trimming the adapter AACTC, cutadapt searches for its colorspace-encoded version 0122. But also TTGAG, CCAGA and GGTCT have an encoding of 0122. This means that effectively four different adapter sequences are searched and trimmed at the same time. There is no way around this, unless the decoded sequence were available, but that is usually only the case after read mapping.

The effect should usually be quite small. The number of false positives is multiplied by four, but with a sufficiently large overlap (3 or 4 is already enough), this is still only around 0.2 bases lost per read on average. If inspecting k-mer frequencies or using small overlaps, you need to be aware of the effect, however.

Double-encoding, BWA and MAQ

The read mappers MAQ and BWA (and possibly others) need their colorspace input reads to be in a so-called "double encoding". This simply means that they cannot deal with the characters 0, 1, 2, 3 in the reads, but require that the letters A, C, G, T be used for colors. For example, the colorspace sequence 0011321 would be AACCTGC in double-encoded form. This is not the same as conversion to basespace! The read is still in colorspace, only letters are used instead of digits. If that sounds confusing, that is because it is.

Note that MAQ is unmaintained and should not be used in new projects.

BWA’s colorspace support was dropped in versions more recent than 0.5.9, but that version works well.

When you want to trim reads that will be mapped with BWA or MAQ, you can use the --bwa option, which enables colorspace mode (-c), double-encoding (-d), primer trimming (-t), all of which are required for BWA, in addition to some other useful options.

The --maq option is an alias for --bwa.

Colorspace examples

To cut an adapter from SOLiD data given in solid.csfasta and solid.qual, to produce MAQ- and BWA-compatible output, allow the default of 10% errors and write the resulting FASTQ file to output.fastq:

cutadapt --bwa -a CGCCTTGGCCGTACAGCAG solid.csfasta solid.qual > output.fastq

Instead of redirecting standard output with >, the -o option can be used. This also shows that you can give the adapter in colorspace and how to use a different error rate:

cutadapt --bwa -e 0.15 -a 330201030313112312 -o output.fastq solid.csfasta solid.qual

This does the same as above, but produces BFAST-compatible output, strips the _F3 suffix from read names and adds the prefix "abc:" to them:

cutadapt -c -e 0.15 -a 330201030313112312 -x abc: --strip-f3 solid.csfasta solid.qual > output.fastq

Bowtie

Quality values of colorspace reads are sometimes negative. Bowtie gets confused and prints this message:

Encountered a space parsing the quality string for read xyz

BWA also has a problem with such data. Cutadapt therefore converts negative quality values to zero in colorspace data. Use the option --no-zero-cap to turn this off.

Sequence Read Archive

The Sequence Read Archive provides files in a special "SRA" file format. When the fastq-dump program from the sra-toolkit package is used to convert these .sra files to FASTQ format, colorspace reads will get an extra quality value in the beginning of each read. You may get an error like this:

cutadapt: error: In read named 'xyz': length of colorspace quality
sequence (36) and length of read (35) do not match (primer is: 'T')

To make cutadapt ignore the extra quality base, add --format=sra-fastq to your command-line, as in this example:

cutadapt -c --format=sra-fastq -a CGCCTTGGCCG sra.fastq > trimmed.fastq

When you use --format=sra-fastq, the spurious quality value will be removed from all reads in the file.