Trimmomatic seems not working

Question

[salmon]

Hello,

I am using paired end RNA sequence for transcriptomic analysis.

java -jar trimmomatic-0.39.jar PE Data/K1_R1.fastq Data/K1_R2.fastq K1_R1_paired.fastq K1_R1_unpaired.fastq K1_R2_paired.fastq K1_R2_unpaired.fastq ILLUMINACLIP:TruSeq3-PE.fa:2:30:10:2:True LEADING:3 TRAILING:3 MINLEN:36

I am getting following result:

Quality encoding detected as phred33
Input Read Pairs: 35521765 Both Surviving: 35521765 (100.00%) Forward Only Surviving: 0 (0.00%) Reverse Only Surviving: 0 (0.00%) Dropped: 0 (0.00%)
TrimmomaticPE: Completed successfully

After running fastqc, I dont find any trimming or shortening of sequence. I even tried by changing the number from 15 to 30 (LEADING:30 TRAILING:30 MINLEN:36) so that quality score is minimum 30 for all the reads but I cannot any difference after running fastqc.

Any help really appreciated.

[Ram]

Does pre-trim FASTQC show bases with quality scores <30?

[cpad0112]

Could you post few example reads from R1 and R2 here? Do not post data images.

[salmon]

Reads from R1

@A00405:433:HHYFCDSX2:3:1101:4815:1000 1:N:0:CCTTGGAA+GAGACGAT
GNAAGACAGAGGTGAAATCCCCGGGCTCAACCTGGGAACTGCCTTTGTGACTGCATAGCTAGAGTACGGTAGAGGGGGAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTTGGAAATCTCGTATGCCGTCTTCGGCTTGTAATGGG
+
F#FFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFF:FFFFFFFF::FF,FFFFFF:F,F,F:F:F,:,,,,,
@A00405:433:HHYFCDSX2:3:1101:7365:1000 1:N:0:CCTTGGAA+GAGACGAT
TNTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTACGTTACTGAGAAGAAACCCTCCCAACAACCAGTTGACATAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTTGGAAATCGCGTATGCCG
+
F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,:FF:FFF,F
@A00405:433:HHYFCDSX2:3:1101:8775:1000 1:N:0:CCTTGGAA+GAGACGAT
GNCATCCATTTTCTTTTTGGAAAAAGGCGCTTGAGGTGGCCGGGAAACCCGGGTCTCAAGCGCCTTTTCTTCGTTCTGATCGGGTGCATCGGGCATCGCCCTCCCAAGCGTTGACCCGATGCGTACTGCCCCCTCTCAGAGATCGGAAGAG

Reads from R2

@A00405:433:HHYFCDSX2:3:1101:4815:1000 2:N:0:CCTTGGAA+GAGACGAT
CCCCCTCTACCGTACTCTAGCTATGCAGTCACAAAGGCAGTTCCCAGGTTGAGCCCGGGGATTTCACCTCTGTCTTGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTGAGACGAGGTGTAGATCTCGGTGGTCGCCGTATCATTAAA
+
FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:F,:FFFF:FFFFFFFFFFFFFF,F:F:FF:FFFFFFFFFFFFFFFFF:::,FFFF
@A00405:433:HHYFCDSX2:3:1101:7365:1000 2:N:0:CCTTGGAA+GAGACGAT
ATGTCAACTGGTTGTTGGGAGGGTTTCTTCTCAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTGAGACGATGTGTAGATCTCGGT
+
FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFF
@A00405:433:HHYFCDSX2:3:1101:8775:1000 2:N:0:CCTTGGAA+GAGACGAT
CTGAGAGGGGGCAGTACGCATCGGGTCAACGCTTGGGAGGGCGATGCCCGATGCACCCGATCAGAACGAAGAAAAGGCGCTTGAGACCCGGGTTTCCCGGCCACCTCAAGCGCCTTTTTCCAAAAAGAAAATGGATGCCAGATCGGAAGAG

[cpad0112]

it's working fine to me.

$ seqkit stats *.f{astq,q}

file                  format  type  num_seqs  sum_len  min_len  avg_len  max_len
K1_R1_paired.fastq    FASTQ   DNA          2      174       78       87       96
K1_R1_unpaired.fastq                       0        0        0        0        0
K1_R2_paired.fastq    FASTQ   DNA          2      174       78       87       96
K1_R2_unpaired.fastq                       0        0        0        0        0
test_R1.fq            FASTQ   DNA          2      302      151      151      151
test_R2.fq            FASTQ   DNA          2      302      151      151      151

Command I used (ubuntu 21.10):

$ TrimmomaticPE test_R1.fq test_R2.fq K1_R1_paired.fastq K1_R1_unpaired.fastq K1_R2_paired.fastq K1_R2_unpaired.fastq ILLUMINACLIP:TruSeq3-PE.fa:2:30:10:2:True LEADING:3 TRAILING:3 MINLEN:36

input R1:

$ cat test_R1.fq 

@A00405:433:HHYFCDSX2:3:1101:4815:1000 1:N:0:CCTTGGAA+GAGACGAT
GNAAGACAGAGGTGAAATCCCCGGGCTCAACCTGGGAACTGCCTTTGTGACTGCATAGCTAGAGTACGGTAGAGGGGGAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTTGGAAATCTCGTATGCCGTCTTCGGCTTGTAATGGG
+
F#FFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFF:FFFFFFFF::FF,FFFFFF:F,F,F:F:F,:,,,,,
@A00405:433:HHYFCDSX2:3:1101:7365:1000 1:N:0:CCTTGGAA+GAGACGAT
TNTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTACGTTACTGAGAAGAAACCCTCCCAACAACCAGTTGACATAGATCGGAAGAGCACACGTCTGAACTCCAGTCACCCTTGGAAATCGCGTATGCCG
+
F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,:FF:FFF,F

Trimmed R1:

$ cat K1_R1_paired.fastq 

@A00405:433:HHYFCDSX2:3:1101:4815:1000 1:N:0:CCTTGGAA+GAGACGAT
GNAAGACAGAGGTGAAATCCCCGGGCTCAACCTGGGAACTGCCTTTGTGACTGCATAGCTAGAGTACGGTAGAGGGGG
+
F#FFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
@A00405:433:HHYFCDSX2:3:1101:7365:1000 1:N:0:CCTTGGAA+GAGACGAT
TNTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTACGTTACTGAGAAGAAACCCTCCCAACAACCAGTTGACAT
+
F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF

Answer 1

[Jeremy Leipzig]

As a sanity check, replace part of 10 or so of the sequences with TACACTCTTTCCCTACACGACGCTCTTCCGATCT (for /1) and GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (for /2)

What does it report now?