adapter trimming miRNA sequencing

Palgrave · 10-03-2014, 05:02 AM

Below is the output from FastQC. Any idea what to trim for? Tried the Illumina Single End Adapter 1 but got no mapping. What are the A, and how do I get rid of them?

ATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAA 4805262 44.91006094070466 Illumina Single End Adapter 1 (95% over 24bp)
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAA 1107781 10.353340196425243 No Hit
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAACA 200679 1.8755493705691118 No Hit
CGATCATCTCGTATGCCGTCTTCTGCTTGAAAAAAA 144914 1.3543687256098158 Illumina PCR Primer Index 7 (96% over 29bp)
TAGCTTATCAGACTGATGTTGACATCTCGTATGCCG 140931 1.3171435394021074 No Hit
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAACAA 124510 1.1636725921972906 No Hit
TGTAAACATCCTTGACTGGAAGCATCTCGTATGCCG 114871 1.0735863331322382 No Hit
TCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAA 112198 1.048604429357896 Illumina Single End Adapter 1 (100% over 21bp)
TCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAA 110008 1.0281366518547872 Illumina Single End Adapter 1 (95% over 23bp)
ATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAC 94442 0.8826565492915952 Illumina Single End Adapter 1 (95% over 24bp)
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAC 82271 0.7689061748667841 No Hit
TGAGGTAGTAGGTTGTATAGTTATCTCGTATGCCGT 77257 0.7220452450035024 No Hit
GTAGTGTTTCCTACTTTATGGAATCTCGTATGCCGT 62069 0.5800979369134498 No Hit
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAACAAA 58659 0.5482280185181984 No Hit

amitm · 10-03-2014, 05:56 AM

Hi Palgrave,
Have you visited here -
http://support.illumina.com/download...ce-letter.html
The pdf file enlists all adapter sequences. If you copy the first entry in your list (without the polyA tail) and search in the pdf, you will see that its the TruSeq adapter.

I wouldn't bother about the polyA and just give the remaining sequence as a contaminant to any adapter trimmer tool. When the tool chops, the polyA's get removed too. I use Trimmomatic (can use multiple threads).

mgogol · 10-03-2014, 12:17 PM

I've used trimmomatic with the adapters file that comes along with fastqc converted to a fasta file. Worked good. The As might be after it reads all the way through the adapter and isn't reading anything, maybe?

relipmoc · 10-03-2014, 08:51 PM

Quote:

Originally Posted by Palgrave

Below is the output from FastQC. Any idea what to trim for? Tried the Illumina Single End Adapter 1 but got no mapping. What are the A, and how do I get rid of them?

ATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAA 4805262 44.91006094070466 Illumina Single End Adapter 1 (95% over 24bp)
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAA 1107781 10.353340196425243 No Hit
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAACA 200679 1.8755493705691118 No Hit
CGATCATCTCGTATGCCGTCTTCTGCTTGAAAAAAA 144914 1.3543687256098158 Illumina PCR Primer Index 7 (96% over 29bp)
TAGCTTATCAGACTGATGTTGACATCTCGTATGCCG 140931 1.3171435394021074 No Hit
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAACAA 124510 1.1636725921972906 No Hit
TGTAAACATCCTTGACTGGAAGCATCTCGTATGCCG 114871 1.0735863331322382 No Hit
TCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAA 112198 1.048604429357896 Illumina Single End Adapter 1 (100% over 21bp)
TCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAA 110008 1.0281366518547872 Illumina Single End Adapter 1 (95% over 23bp)
ATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAC 94442 0.8826565492915952 Illumina Single End Adapter 1 (95% over 24bp)
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAC 82271 0.7689061748667841 No Hit
TGAGGTAGTAGGTTGTATAGTTATCTCGTATGCCGT 77257 0.7220452450035024 No Hit
GTAGTGTTTCCTACTTTATGGAATCTCGTATGCCGT 62069 0.5800979369134498 No Hit
GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAACAAA 58659 0.5482280185181984 No Hit

Hi Palgrave,

From the pdf file provided by illimina, you will find ATCTCGTATGCCGTCTTCTGCTTG is actually the v1.5 Small RNA 3' Adapter. Never mind those tailing poly-A as they are fake base-calls by the illumina sequencer if there is no base detected. A mature adapter trimmer will trim those tailing poly-A next to adapter sequence.

Though trimmomatic seems to be specifically optimized for small RNA adapter trimming, I also recommend you using the skewer (http://sourceforge.net/projects/skewer ) tool which is not optimized for any specific applications but performs equally well or even better for small RNA adapter trimming.

For your case, the command is very simple like this:

Code:

skewer -x ATCTCGTATGCCGTCTTCTGCTTG -l 16 -L 30 yourData.fastq

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10-03-2014, 05:02 AM	#1
Palgrave Member Location: norway Join Date: Aug 2011 Posts: 73	adapter trimming miRNA sequencing Below is the output from FastQC. Any idea what to trim for? Tried the Illumina Single End Adapter 1 but got no mapping. What are the A, and how do I get rid of them? ATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAA 4805262 44.91006094070466 Illumina Single End Adapter 1 (95% over 24bp) GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAA 1107781 10.353340196425243 No Hit GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAACA 200679 1.8755493705691118 No Hit CGATCATCTCGTATGCCGTCTTCTGCTTGAAAAAAA 144914 1.3543687256098158 Illumina PCR Primer Index 7 (96% over 29bp) TAGCTTATCAGACTGATGTTGACATCTCGTATGCCG 140931 1.3171435394021074 No Hit GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAACAA 124510 1.1636725921972906 No Hit TGTAAACATCCTTGACTGGAAGCATCTCGTATGCCG 114871 1.0735863331322382 No Hit TCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAA 112198 1.048604429357896 Illumina Single End Adapter 1 (100% over 21bp) TCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAA 110008 1.0281366518547872 Illumina Single End Adapter 1 (95% over 23bp) ATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAC 94442 0.8826565492915952 Illumina Single End Adapter 1 (95% over 24bp) GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAC 82271 0.7689061748667841 No Hit TGAGGTAGTAGGTTGTATAGTTATCTCGTATGCCGT 77257 0.7220452450035024 No Hit GTAGTGTTTCCTACTTTATGGAATCTCGTATGCCGT 62069 0.5800979369134498 No Hit GTATGCCGTCTTCTGCTTGAAAAAAAAAAAAACAAA 58659 0.5482280185181984 No Hit

10-03-2014, 05:56 AM	#2
amitm Member Location: Manchester, UK Join Date: Feb 2011 Posts: 52	Hi Palgrave, Have you visited here - http://support.illumina.com/download...ce-letter.html The pdf file enlists all adapter sequences. If you copy the first entry in your list (without the polyA tail) and search in the pdf, you will see that its the TruSeq adapter. I wouldn't bother about the polyA and just give the remaining sequence as a contaminant to any adapter trimmer tool. When the tool chops, the polyA's get removed too. I use Trimmomatic (can use multiple threads).

10-03-2014, 12:17 PM	#3
mgogol Senior Member Location: Kansas City Join Date: Mar 2008 Posts: 197	I've used trimmomatic with the adapters file that comes along with fastqc converted to a fasta file. Worked good. The As might be after it reads all the way through the adapter and isn't reading anything, maybe?