trimming with cutadapt

Question

[mgadrianam]

Hi, everyone

I have libraries of microRNAs, I do the trimming with cutadapt but It report different results

This is the raw data using FastQC

Filename    SRR837437.fastq.gz
File type    Conventional base calls
Encoding    Sanger / Illumina 1.9
Total Sequences    17830678
Filtered Sequences    0
Sequence length    50
%GC    57

Basic statistics are ok, per base sequence quality ok, per sequence quality scores are ok, per base sequence content not ok, per base GC not ok, per base N content ok, Sequence Length Distribution ok, Sequence Duplication Levels not ok, overrepresented sequences

TCCTGTACTGAGCTGCCCCGAGTGGAATTCTCGGGTGCCAAGGAACTCCA    6084670    34.124725935828124    RNA PCR Primer, Index 1 (100% over 28bp)
TCCTGTACTGAGCTGCCCCGATGGAATTCTCGGGTGCCAAGGAACTCCAG    5105541    28.63346531186307    RNA PCR Primer, Index 1 (100% over 29bp)
TCCTGTACTGAGCTGCCCCGAGATGGAATTCTCGGGTGCCAAGGAACTCC    1847951    10.363885209524842    RNA PCR Primer, Index 1 (100% over 27bp)
TCCTGTACTGAGCTGCCCCGTGGAATTCTCGGGTGCCAAGGAACTCCAGT    509674    2.858410656061424    RNA PCR Primer, Index 1 (100% over 30bp)
TCCTGTACTGAGCTGCCCCGAGTTGGAATTCTCGGGTGCCAAGGAACTCC    484975    2.719890965447304    RNA PCR Primer, Index 1 (100% over 27bp)
TATTGCACTTGTCCCGGCCTGTTGGAATTCTCGGGTGCCAAGGAACTCCA    143730    0.806082640267521    RNA PCR Primer, Index 1 (100% over 28bp)
TCCTGTACTGAGCTGCCCCGATGGAATTCTCGGGGGCCAAGGAACTCCAG    121167    0.6795423034390503    RNA PCR Primer, Index 1 (96% over 29bp)
TATTGCACTTGTCCCGGCCTGTGGAATTCTCGGGTGCCAAGGAACTCCAG    114725    0.6434135594843898    RNA PCR Primer, Index 1 (100% over 29bp)
TCCTGTACTGAGCTGCCCCGAGGGGAATTCTCGGGTGCCAAGGAACTCCA    112651    0.631781921023979    RNA PCR Primer, Index 1 (96% over 28bp)
TCCTGTACTGAGCTGCCCCGAATGGAATTCTCGGGTGCCAAGGAACTCCA    111827    0.6271606721853201    RNA PCR Primer, Index 1 (100% over 28bp)
TCCTGTACTGAGCTGCCCCGAGGGGAATTCTCGGGGGCCAAGGAACTCCA    102622    0.5755361630107392    No Hit
TCCTGTACTGAGCTGCCCCGAGTGGAATTCTCGGGGGCCAAGGAACTCCA    83983    0.4710028412828722    RNA PCR Primer, Index 1 (96% over 28bp)
TCCTGTACTGAGCTGCCCCGGTGGAATTCTCGGGTGCCAAGGAACTCCAG    68349    0.3833224962056967    RNA PCR Primer, Index 1 (100% over 29bp)
TATTGCACTTGTCCCGGCCTGTATGGAATTCTCGGGTGCCAAGGAACTCC    52227    0.29290529502018936    RNA PCR Primer, Index 1 (100% over 27bp)
TCCTGTACTGAGCTGCCCCGAGATGGAATTCTCGGGGGCCAAGGAACTCC    50813    0.28497514228006365    RNA PCR Primer, Index 1 (96% over 27bp)
TATTGCACTTGTCCCGGCCTGATGGAATTCTCGGGTGCCAAGGAACTCCA    41374    0.2320382881682906    RNA PCR Primer, Index 1 (100% over 28bp)
CAACGGAATCCCAAAAGCAGCTGTGGAATTCTCGGGTGCCAAGGAACTCC    35506    0.19912871512793848    RNA PCR Primer, Index 1 (100% over 27bp)
TCCTGTACTGAGCTGCCCCGAGAATGGAATTCTCGGGTGCCAAGGAACTC    27244    0.1527928438840071    RNA PCR Primer, Index 1 (100% over 26bp)
TCCTGTACTGAGCTGCCCCGATTGGAATTCTCGGGTGCCAAGGAACTCCA    26773    0.150151329074531    RNA PCR Primer, Index 1 (100% over 28bp)
TATTGCACTTGTCCCGGCCTGTTTGGAATTCTCGGGTGCCAAGGAACTCC    23100    0.12955200020997518    RNA PCR Primer, Index 1 (100% over 27bp)
TCCTGTACTGAGCTGCCCTGGAATTCTCGGGTGCCAAGGAACTCCAGTCA    20684    0.11600231914905311    RNA PCR Primer, Index 1 (100% over 32bp)
TCCTGTACTGAGCTGCCCCGAGATTGGAATTCTCGGGTGCCAAGGAACTC    20448    0.11467875758846635    RNA PCR Primer, Index 1 (100% over 26bp)
AAACCGTTACCATTACTGAGTTGGAATTCTCGGGTGCCAAGGAACTCCAG    19780    0.11093240537460214    RNA PCR Primer, Index 1 (100% over 29bp)
TTCCTGTACTGAGCTGCCCCGATGGAATTCTCGGGTGCCAAGGAACTCCA    19030    0.10672617160155098    RNA PCR Primer, Index 1 (100% over 28bp)
CCACGTTCCCGTGGTGGAATTCTCGGGTGCCAAGGAACTCCAGTCACCGA    18687    0.10480252068934227    RNA PCR Primer, Index 2 (100% over 36bp)
TCCTGTACTGAGCTGCCCCGGGTGGAATTCTCGGGTGCCAAGGAACTCCA    18033    0.10113468483924166    RNA PCR Primer, Index 1 (100% over 28bp)

I run cutadapt

cutadapt -a GGAATTCTCGGGTGCCAAGG SRR837437.fastq.gz -m 17 --length-tag 'length=' -o SRR837437.cutadapt1.fastq

The basis statistic are

Filename    SRR837437.cutadapt1.fastq
File type    Conventional base calls
Encoding    Sanger / Illumina 1.9
Total Sequences    17698809
Filtered Sequences    0
Sequence length    17-50
%GC    59

change Per base sequence quality to not ok

and the overrepresented sequences are

TCCTGTACTGAGCTGCCCCGAGT      6470528    36.55911536194328    No Hit
TCCTGTACTGAGCTGCCCCGAT         5532068    31.25672467565473    No Hit
TCCTGTACTGAGCTGCCCCGAGAT    2005215    11.329660656827247    No Hit
TCCTGTACTGAGCTGCCCCGT           570466    3.2231886337662607    No Hit
TCCTGTACTGAGCTGCCCCGAGTT    530257    2.996003855400666    No Hit
TCCTGTACTGAGCTGCCCCGAGG      317138    1.7918606839590168    No Hit
TATTGCACTTGTCCCGGCCTGTT        151241    0.8545264260436959    No Hit
TCCTGTACTGAGCTGCCCCGAAT        121369    0.6857467075891943    No Hit
TATTGCACTTGTCCCGGCCTGT           120442    0.6805090670225323    No Hit
TCCTGTACTGAGCTGCCCCGGT          72406    0.4091009739694914    No Hit
TATTGCACTTGTCCCGGCCTGTAT        54738    0.30927504782948956    No Hit
TATTGCACTTGTCCCGGCCTGAT          43588    0.24627645848938196    No Hit
CAACGGAATCCCAAAAGCAGCTGT      37011    0.20911576592526648    No Hit
TCCTGTACTGAGCTGCCCCGAGAAT    30626    0.1730398921192946    No Hit
TTCCTGTACTGAGCTGCCCCGAT         29613    0.1673163431505476    No Hit
TCCTGTACTGAGCTGCCCCGATT         29239    0.1652032066112471    No Hit
TCCTGTACTGAGCTGCCCCGAGAG     28483    0.16093173275105688    No Hit
TATTGCACTTGTCCCGGCCTGTTT       24326    0.13744427661770914    No Hit
TCCTGTACTGAGCTGCCCCGAG          23792    0.13442712444662236    No Hit
TCCTGTACTGAGCTGCCCT                  22984    0.12986184550610155    No Hit
TCCTGTACTGAGCTGCCCCGAGATT    22301    0.1260028287779138    No Hit
TTCCTGTACTGAGCTGCCCCGAGT    21007    0.11869160235584214    No Hit
TCCTGTACTGAGCTGCCCCGAGGT    20634    0.11658411591424034    No Hit
AAACCGTTACCATTACTGAGTT    20628    0.11655021532804835    No Hit
TCCTGTACTGAGCTGCCCCGGGT    19676    0.11117132231891988    No Hit
TCCTGTACTGAGCTGCCCCT    19513    0.11025035639403759    No Hit
TCCTGTACTGAGCTGCCCCGAGA    19409    0.1096627462333765    No Hit

because I have several file I use

for i in *fastq.gz;
do
  echo $i;
  cutadapt -a TGGAATTCTCGGGTGCCAAGG -m 17 -o ${i/.fastq/}.cutadapt.fastq --length-tag 'length=' $i;
done

and show the basic statistics

Filename    SRR837437.gz.cutadapt.fastq
File type    Conventional base calls
Encoding    Sanger / Illumina 1.9
Total Sequences    17683138
Filtered Sequences    0
Sequence length    17-50
%GC    62

the show also change not ok of Per base sequence quality and the data before cutadapt was ok and the overrepresented sequences

TCCTGTACTGAGCTGCCCCGAG    6766671    38.26623419440599    No Hit
TCCTGTACTGAGCTGCCCCGA    5552096    31.3976851846092    No Hit
TCCTGTACTGAGCTGCCCCGAGA    2029103    11.474790277607967    No Hit
TCCTGTACTGAGCTGCCCCG    574490    3.2488012025919835    No Hit
TCCTGTACTGAGCTGCCCCGAGT    534294    3.0214886068298514    No Hit
TATTGCACTTGTCCCGGCCTGT    151437    0.8563921177338547    No Hit
TCCTGTACTGAGCTGCCCCGAA    121949    0.6896343850282682    No Hit
TATTGCACTTGTCCCGGCCTG    120821    0.6832554267234695    No Hit
TCCTGTACTGAGCTGCCCCGG    74513    0.4213788299339178    No Hit
TATTGCACTTGTCCCGGCCTGTA    54785    0.30981492085850376    No Hit
TATTGCACTTGTCCCGGCCTGA    43643    0.24680574228397698    No Hit
CAACGGAATCCCAAAAGCAGCTG    37189    0.21030769538754943    No Hit
TCCTGTACTGAGCTGCCCCGAGAA    30837    0.17438646918889622    No Hit
TCCTGTACTGAGCTGCCCCGAT    30562    0.17283131534685756    No Hit
TTCCTGTACTGAGCTGCCCCGA    30326    0.17149671059514437    No Hit
TTCCTGTACTGAGCTGCCCCGAG    27068    0.15307237889564623    No Hit
TATTGCACTTGTCCCGGCCTGTT    24360    0.13775835488022545    No Hit
TCCTGTACTGAGCTGCCC    23470    0.13272531153690031    No Hit
TCCTGTACTGAGCTGCCCCGAGG    22859    0.12927004245513438    No Hit
TCCTGTACTGAGCTGCCCCGAGAT    22565    0.12760744162037305    No Hit
TCCTGTACTGAGCTGCCCCGGG    21446    0.1212793792594957    No Hit
AAACCGTTACCATTACTGAGT    20642    0.11673267493586263    No Hit
TCCTGTACTGAGCTGCCCC    20219    0.11434056557156315    No Hit

Why it is different it is the same command, it is necessary to clean all this overrepresented sequences and why decrease the per base sequence quality?

Thanks for your help

Answer 1

[GouthamAtla]

No need to clean over represented sequences if they are not a part of any index sequences.

Regarding different output, why don't you use parallel and just do dry-run and see if the commands your are running are exactly same.

parallel --dry-run cutadapt -a TGGAATTCTCGGGTGCCAAGG -m 17 -o cutadapt_{.} --length-tag \'length=\' {} ::: *.fastq.gz​

[mgadrianam]

Hi, Is it normal that after the trimming of adapter the quality per base sequence decrease? I use cutadapt before and it did not decrease the quality per base sequence.

Thanks you so much

Adriana