Remove cDNA synthesis adaptors

Quality check of the raw data

Results of the quality check of the raw data can be used as a reference point to check the improvements done by this step.

mkdir $OUT
fastqc --outdir=$OUT --noextract --threads=$CPUS $IN/*.fastq

Split the files by multiplex tags

If you used multiplexing during library preparation, your reads have either already been split in your sequencing facility, or you have to split the reads according to the ‘barcodes’ stored in the sequences yourself.

  • for 454, this is done by sfffile, you have to convert resulting sff files to fastq format
  • for Illumina this can be done e.g. by eautils
mkdir -p $OUT

parallel -j 1 sfffile -s RLMIDs -o $OUT/{/.} {} ::: $IN/*.sff

Remove cDNA synthesis primers with cutadapt

Another source of noise in the data is the primers that were used for reverse transcription of mRNA and for the following PCR amplification of cDNA. We remove them using cutadapt.

If you have more or less than three primers, you have to change the command by adding/removing --anywhere= parts.

# data from previous steps
mkdir $OUT

# cut out the evrogen sequences using GNU parallel and cutadapt
# cutadapt supports only 'N' wildcards, no ambiguity codes
parallel -j $CPUS "cutadapt --anywhere=$PRIMER1 --anywhere=$PRIMER2 --anywhere=$PRIMER3 \
  --error-rate=0.2 --overlap=15 --minimum-length=40 \
  --output=$OUT/{/.}.fastq --rest-file=$OUT/{/.}.rest {}" ::: $IN/*.fastq > $OUT/cutadapt.log

Check the results

It is necessary to check the results of adaptor cutting.

First you can check how many of the primers were missed by cutadapt. agrep uses a different matching algorithm than cutadapt, so some remaining hits are usually found. /dev/null is used as the second input to agrep so the filenames are output.

parallel agrep -c -$NERR "$PRIMER3" {} /dev/null ::: $OUT/*.fastq|grep -v /dev

The next thing to check is the logs produced by cutadapt. Results for our data - 454 Titanium data from Smart kit synthesized cDNA:

  • ~70% trimmed reads
  • ~10% trimmed basepairs
  • ~10% too short reads
grep -A5 Processed $OUT/cutadapt.log | less

The mean length of the removed sequences should be close to length of the adapter (31 in this case):

less $OUT/cutadapt.log
# Lengths of removed sequences (5')
# length  count   expected
# 5       350     264.7
# 6       146     66.2
# ...
# 30      6414    0.0
# 31      63398   0.0
# 32      6656    0.0
# ...

The size of the .rest files is 1/500 of the .fastq (should be 1/250 for .fasta)

ls -l $OUT

The fastqc checks should be +- ok.

fastqc --outdir=13-fastqc --noextract --threads=8 $OUT/*.fastq

Visual debugging

If something in the previous checks looks weird, look directly at the data. Substitute filenames below with the names of your files.

Look where the primers are in the sequence. tre-agrep is used to color the output of agrep, because agrep throughput is ~ 42 MB/s while tre-agrep throughput is ~ 2 MB/s.

agrep -n -$NERR "$PRIMER3" $FQFILE |tre-agrep -$NERR "$PRIMER3" --color|less -S -R

To find out how many differences should be allowed in the pattern matching, we try to find a value of NERR where the primer sequence starts to match randomly inside the reads, and not only in at the beginning. Notice the ^ in the first command, marking the start of the read.

agrep -c -$NERR "^$PRIMER3" $FQFILE && agrep -c -$NERR "$PRIMER3" $FQFILE

# numbers for tag-cleaned G59B..
# 4 errors: 11971 12767
# 5 errors: 16366 17566
# 6 errors: 17146 23858
# 7 errors: 18041 67844

In our sample results, numbers start to diverge for NERR > 5, so 5 is a good choice.

Read count statistics

For a single file:

# read count statistics
# @ can be in the beginning of quality string, so filter the rows in order

# count of sequences
awk '((NR%4)  == 1)' $FQFILE | wc -l
# or more effective
echo $(( $(wc -l $FQFILE) / 4 ))

# count of sequenced bases
awk '((NR%4)  == 2)' $FQFILE | wc -m

For all files in OUT:

# parallel, IO bound task, so run one process a time
echo "read_count base_count filename"
parallel -j 1 'echo $( gawk "((NR%4)  == 1)" {} | wc -l ) $( gawk "((NR%4)  == 2)" {} | wc -m ) {}' ::: $OUT/*.fastq