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I think the easiest thing to do would be to filter out low-quality reads and then run FastQC on the high-quality reads to see if they have any problems. This is a bit tangential to the topic, but for example you can remove low-quality reads with BBDuk using the maq (min average quality) flag, like "maq=15".
On a more thread-related note, the BBMap package has all of the Illumina adapter sequences in a fasta file which is more convenient than Illumina's PDF format. It's in /bbmap/resources/adapters.fa once you unzip/untar the package. As pmiguel indicated, this is just adapters; there are a lot of other sequences that Illumina considers proprietary and forbids sharing.
I'd also like to note that if you have paired-end reads with an unknown adapter sequence, you can discover it with BBMerge like this:
bbmerge.sh in1=r1.fq in2=r2.fq outa=adapters.fa
This tends to be useful in situations like discovering PhiX adapters, which are not in Illumina's customer letter.Last edited by Brian Bushnell; 05-28-2016, 07:57 AM.
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Hi everyone!
Does anyone knows if/how is ir possible to distinguish kmer plots in FASTQC of reads derived from RRBS librarys from reads that just have bad quality?thanks in advance
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The document (yes, you have the latest version) lists all of the adapter sequences ever offered by Illumina (or Nextera, whose technology it acquired). The methods have evolved, from single-end to paired-end sequencing, and unindexed to single indexing to dual indexing. All of those changes have required changes in the adapter sequences (and sequencing/indexing primers). And Illumina does offer a separate document that indicates which of their library prep versions are or are not compatible with which sequencing kits/versions.
Publications are a lagging indicator, so the sequences in a paper may be obsolescent by the time of release.
I explicitly stated which adapters are compatible with the current version of sequencing. Your service provider should be able to verify this information for you, and/or assist you with primer design.
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Originally posted by HESmith View PostThe sequences that you list are from the earliest version of Illumina multiplexing, which was supplanted by the TruSeq platform several years ago. If you want your adapter sequences to be compatible with the current generation of sequencing and index primers, download the Customer Sequence Letter from Illumina and use the TruSeq v1/v2 (pp. 12-13) Universal Adapter plus the reverse complement of the Truseq Indexed Adapter (plus 3' A) to design your amplicon primers. If you require dual indexing, use the TruSeq HT sequences (D500 + D700 rev comp, pp. 10-11) as the basis of your design.
The sequences I listed that are labeled for multiplexing are the same as those listed for TruSeq, so I've been tempted to go with those. However, I can't be sure because there are so many different sequences out there. Both the Nextera and small RNA sample prep kits use still different sequences. Furthermore, a recent publication (Nature Methods, published last year) from which I and some colleagues are trying to replicate some methods used oligos that match the PE sequences listed earlier, not the TruSeq sequences. It seems that Illumina is still using a few different adapter sequences, and I don't understand how that can be.
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Originally posted by ajthomas View PostWhich is the correct sequence?
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Originally posted by ssully View PostI'm quite lost. I've got a set of paired end 100 nt HiSeq reads (library insert size was ~300bp) from a colleague and I can't determine what adapter sequences to trim. The current Illumina letter to customers lists a variety of sequences. Can someone explain the use of this set
versus this set?
For a paired end set that includes indices (barcodes) which set would have been used?
Also, what are the 2 sequences of the oligos that coat a paired end flowcell used in the current HiSeq platform? (these would be sequences complementary to adapters sequences)
The confusion comes from these adapter sequences:
PE PCR Primer 2.0
5' CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
PE Read 2 Sequencing Primer
5' CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
Multiplexing PCR Primer 2.0
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
Multiplexing Read 2 Sequencing Primer
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
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(reply to clueheart)
Just wanna remind you:
HP11 combines at least 2 kinds of sequencing primer.
The sequence you posted should be one of them, but I dont know the exact letter too, maybe no one knows except Illumina.
btw, the other primer should fit NextEra system.
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Hi,
I'm following the protocol from this TCR sequencing paper: http://www.nature.com/nbt/journal/v3.../nbt.2938.html
The PE primers they used are:
PEprimerl AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
PEprimer2 AAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
Should I tell sequencing center to use P5 and P7 primers for MiSeq for the PCR product? I can see P5 primer sequence in PEprimer1 but PEprimer2 doesn't have exact P7 sequence. It lacks first "C"? Could anyone help me?
Thank you!
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Originally posted by clueheart View PostHi,
i am uncertain what is the actual sequence of the read2 primer on Hiseq, PE amplicon sequencing.
To my understanding it should be HP11.
Is ATCTCGTATGCCGTCTTCTGCTTG the correct sequence for HP11 ?
--
Phillip
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Hi,
i am uncertain what is the actual sequence of the read2 primer on Hiseq, PE amplicon sequencing.
To my understanding it should be HP11.
Is ATCTCGTATGCCGTCTTCTGCTTG the correct sequence for HP11 ?
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Originally posted by ssully View PostIllumina helped sort me out. The seqs I posted last time are from an old , discontinued version of the kit (pp 20-21 of the July 2014 letter). The newer TruSeq adapters('universal' and 'indexed') are found on pp 12-13 of the letter .
These 'floppy end' adapters dimerize only at their last 12 nt. After they're ligated to flanks of the insert DNA fragment the result looks like this:
Code:universal indexed 5' AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT---insert--AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3' ||||||||||||||||||||||||||||||||||||| 3' GTTCGTCTTCTGCCGTATGCTCTAGNNNNNNCACTACACTGACCTCAAGTCTGCACACGAGAAGGCTAGA---insert--TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5' indexed universal
Code:5' AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATC-T-insert-A-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7]ATCTCGTATGCCGTCTTCTGCTTG 3' ||||||||||||||||||||||||||||||||||| 3' GTTCGTCTTCTGCCGTATGCTCTA[i7]CACTGACCTCAAGTCTGCACACGAGAAGGCTAG-A-insert-T-CTAGCCTTCTCGCAGCACATCCCTTTCTCACA[i5]CACATCTAGAGCCACCAGCGGCATAGTAA 5'
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Indexed PCR Primer Ordering
Hello, I'm new here so apologies if this question has been answered.
I'm following the Buckler Lab GBS protocol and only have access to 48 adapters for ApeKI. I'd like to multiplex these into one Illumina lane using indexed PCR primers. I found sequences for PCR primers from the ddRAD protocol(Peterson). Do I have to alter the sequence at all to match my ApeKI adapters? Do I have to request anything particular when ordering these primers from IDT? Or would it be better for me to just order the NEBNext Multiplex Oligos for Illumina Kit? (https://www.neb.com/products/e7335-n...-primers-set-1)
ApeKI Common Adapter:
5'-CWGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
5'-CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
ApeKI Barcoded Adapter:
5’-CWGxxxxAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTyyyy
PCR primers I'm considering ordering:
PCR1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG
PCR2_Idx_1_ATCACG: CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_2_CGATGT: CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_3_TTAGGC: CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGC
PCR2_Idx_4_TGACCA: CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGC
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Illumina helped sort me out. The seqs I posted last time are from an old , discontinued version of the kit (pp 20-21 of the July 2014 letter). The newer TruSeq adapters('universal' and 'indexed') are found on pp 12-13 of the letter .
These 'floppy end' adapters dimerize only at their last 12 nt. After they're ligated to flanks of the insert DNA fragment the result looks like this:
Code:universal indexed 5' AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT---insert--AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3' ||||||||||||||||||||||||||||||||||||| 3' GTTCGTCTTCTGCCGTATGCTCTNNNNNNAGCACTACACTGACCTCAAGTCTGCACACGAGAAGGCTAGA---insert--TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA 5' indexed universal
P1: 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTA
(i.e. the first 44 bases of the universal adapter)
P2: 5' CAAGCAGAAGACGGCATACGAGAT
(reverse complement of the last 24 bases of the indexed primer)
P2 primes first, and then the ssDNA from P2 priming/extension becomes the template for P1. So the PCR product is:
Code:5'AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT---insert-----AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTATGCCGTCTTCTGCTTG 3' 3'TTACTATGCCGCTGGTGGCTCTAGATGTGTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA---insert-----TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGNNNNNNTAGAGCATACGGCAGAAGACGAAC 5'
The enriched PCR product can then be denatured and each strand hydbridized to the bound flowcell oligos for cluster generation and reading.Last edited by ssully; 11-18-2014, 01:57 PM.
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I'm quite lost. I've got a set of paired end 100 nt HiSeq reads (library insert size was ~300bp) from a colleague and I can't determine what adapter sequences to trim. The current Illumina letter to customers lists a variety of sequences. Can someone explain the use of this set
Oligonucleotide sequences for Paired End DNA
PE Adapters
5' P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
PE PCR Primer 1.0
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
PE PCR Primer 2.0
5' CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
PE Read 1 Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
PE Read 2 Sequencing Primer
5' CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT
Oligonucleotide sequences for the Multiplexing Sample Prep Oligo Only Kit
Multiplexing Adapters
5' P-GATCGGAAGAGCACACGTCT
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Multiplexing PCR Primer 1.0
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
Multiplexing PCR Primer 2.0
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
Multiplexing Read 1 Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Multiplexing Index Read Sequencing Primer
5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
Multiplexing Read 2 Sequencing Primer
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
PCR Primer, Index 1
5’ CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTC
PCR Primer, Index 2
5’ CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTC
.
.
.etc for 12 indexes total..
For a paired end set that includes indices (barcodes) which set would have been used?
Also, what are the 2 sequences of the oligos that coat a paired end flowcell used in the current HiSeq platform? (these would be sequences complementary to adapters sequences)Last edited by ssully; 11-17-2014, 07:22 PM.
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