incomplete job

[yaximik]

Martin,

Here is a line of code from my cleaning script:

# remove CL73 adaptor from 3'-ends of read 2 (shown as complement
# to its 3'->5' sequence in read 2
find_adaptor -l 3 -r GGAAGAGCGTCGTGTAGGGAAAGAGTGT |
clip_adaptor |

The scrip executes fine. However, when I aligned cleaned reads to a reference, I found the following (image attached). The two to lines still show the presense of the CL73 adaptor end (shown in the code in bold underscored), which supposed to be removed.
Is anything wrong with my code or what may be the reason from incomplete job? Should I just re-run the script on cleaned data?

Vladimir

[Martin Asser Hansen]

Try to make a test a single read containing a partial adaptor. Remember that -r is 3'-5' and you should use -R for 5'-3' adaptor.

Cheers,

Martin

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[yaximik]

Martin,

I am at complete loss. I created test file and tried to clean it. I observed the same behavior, but I cannot explain why my script removes only some part of adaptors. Here is the script:

#!/bin/sh

perl -pne 'if (($. % 4) == 0) {tr/[_-~]/_/}' | #change all scores in the range "_"(95) to "~"(126) to "_"(95)
read_fastq -e base_33 -i - |         # Read in raw FASTQ data from stdin
mask_seq |                # soft mask quality < 20 in lowercase
trim_seq -m 30 -l 4 |            #trim Q<30 until min good stretch > 4
grab -e 'SEQ_LEN >= 35' |         # Filter out reads < 35 nt
# remove CL78 adaptor from read 1
find_adaptor -L 6 -R AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
clip_adaptor |
grab -e 'SEQ_LEN >= 35' |         # Filter out reads < 35 nt
# remove CL73 adaptor from read 2
find_adaptor -L 6 -R GGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATC |
clip_adaptor |
grab -e 'SEQ_LEN >= 35' |         # Filter out reads < 35 nt
mean_scores |                           # Calculate mean quality score
grab -e "SCORES_MEAN >= 30" |           # Filter reads with score mean < 30
mean_scores -l |                        # Calculate local mean quality score
grab -e "SCORES_MEAN_LOCAL >= 20" |     # Filter reads with local score mean < 20
write_fastq -e base_33 -x               # Write raw FASTQ data to stdout

Here is the test file:

@read 1 full CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBB
@read 1 half CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGGAAGAGCACACGTCNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBB
@read 1 10nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGGAAGNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBB
@read 1 8nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGGANNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBB
@read 1 7nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGGNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBB
@read 1 6nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBB
@read 1 5nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBB
@read 2 33nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCNNNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBBBB
@read 2 20 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGCGTCGTGTAGGGANNNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBBBB
@read 2 10 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGCGTNNNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBBBB
@read 2 7 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGNNNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBBBB
@read 2 6 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGANNNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBBBB
@read 2 5 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGNNNNNNNNNNNN
+
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhBBBBBBBBBBBB

Here is the cleaning result

@read 1 full CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGGAAGAGCACACGTCTGAACTCC
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 1 half CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGGAAGAGCAC
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 1 10nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATC
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 1 8nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGA
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 1 7nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 1 6nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 1 5nt CL78
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 2 33nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGCGTCGTGTAGGGAAAGAGT
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 2 20 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAAGAGCGTC
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 2 10 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAA
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 2 7 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 2 6 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
@read 2 5 nt CL73
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

As you see it removes completely only shorter (partial) adaptors, but only trims 6nt of longer adaptors from their 3' ends, presumably along with NNNN tails.

Please advise - what could be wrong?

[Martin Asser Hansen]

Check this out. I saved your test data to test.fq and did this:

maasha@mel:~$ read_fastq -n 1 -e base_33 -i test.fq | find_adaptor -L 6 -R AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC  | clip_adaptor

SEQ_NAME: read 1 full CL78

SEQ: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATCGGAAGAGCACACGTCTGAACTCC

SEQ_LEN: 67

SCORES: IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

ADAPTOR_POS_RIGHT: 67

ADAPTOR_LEN_RIGHT: 16

ADAPTOR_PAT_RIGHT: AGTCACNNNNNNNNNN

---

maasha@mel:~$ read_fastq -n 1 -e base_33 -i test.fq | find_adaptor -L 6 -r AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC  | clip_adaptor

SEQ_NAME: read 1 full CL78

SEQ: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

SEQ_LEN: 37

SCORES: IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

ADAPTOR_POS_RIGHT: 37

ADAPTOR_LEN_RIGHT: 36

ADAPTOR_PAT_RIGHT: TTAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

---

Notice the difference between -r and -R

And do make sure the different steps in your cleaning script works before putting it all together :o)

Cheers,

Martin