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Canonical ILLUMINA library design as of June 2012 (all 5'-3'), "TruSeq V3": NOTE all sequences shown are TOP STRAND 5' to 3'
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If you'd like a different description, this one from the Tufts core facility is quite good.
NOTE THAT THE SHADED PORTIONS SHOULD NOT BE CHANGED if you are designing your own primers!! The only flexibility one has is in the "template" section and in the two "index read" sections. Every other nucleotide shown matters as-is.
Single index adaptor design on a standard Illumina HiSeq or MiSeq run
Highlight color red P5 PCR primer/flowcell capture site: AATGATACGGCGACCACCGAGA
Highlight color yellow IndexRead2: NONE - as in do NOT put an index here. If you want to add an index here, use one of the "Dual-index" designs below.
Highlight color green Read1 primer site: Either the small RNA sequencing primer site: (NEB: TCTACACGTTCAGAGTTCTACAGTCCGACGATCA [Illumina lists this but it is UNPROVEN: CAGGTTCAGAGTTCTACAGTCCGACGATCA]) OR the standard TruSeq Read 1 primer site: TCTACACTCTTTCCCTACACGACGCTCTTCCGATCT. Which to choose? The TruSeq Read 1 primer site is complementary to the Read 2 primer site, so if you are designing amplicons do NOT use the TruSeq Read 1 primer site, use the small RNA sequencing primer site.
- The insert to be sequenced
Highlight color cyan Read2 primer site: Then the Index read primer site: AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC (NOTE: the initial A is from the dA tailing of the insert and is not included in the index primer or adaptor sequences; note also the reverse-complement of this is the Read 2 sequencing primer, but the Read 2 sequencing primer includes the T corresponding to the dA insert tail so sequencing starts with the insert)
Highlight color blue IndexRead1: The index sequence (usually 6 bp) - see many examples below in the Barcodes section. Within a lane, image analysis works best with as much base diversity as possible.
Highlight color purple P7 PCR primer/flowcell capture site: ATCTCGTATGCCGTCTTCTGCTTG
Here is an example of a read-pair from an RNA-seq library generated from the NEB small RNA kit with an insert size of 62 nt:
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Read 1 sequence (note adaptor sequence starting with "AGATCGGAA...")
AAGGGATCATAGACGGTATTTCTATGTAAACGAACAGTCGGGCGAGTCTCAGTGGGAGTTTCAGATCGGAAGAGCACACGTCTGAACTCCAGNCACCGATG
ACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATCAGGGATCATAGACGGTATTTCTATGTAAACGAACAGTCGGGCGAGTCTCAGTGGGAGTTTC
5' SR adaptor:GUUCAGAGUUCUACAGUCCGACGAUC
Read 2 sequence, reverse complemented (note adaptor sequence RC ending with "...CCGACGATCA")
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Dual-index TruSeq (NOT Nextera) adaptor design on a standard Illumina PE HiSeq or MiSeq run
Highlight color red P5 PCR primer/flowcell capture site: AATGATACGGCGACCACCGAGATCTACAC
Highlight color yellow IndexRead2: Optional. Example: TAGATCGC. This is called "IndexRead2" because it is read after index read 1. The GSAF does not normally sequence this barcode - please request if you need it read. We have little guidance to offer on designs other than to re-use the same sequences as in the Index Read 1 site - base diversity is your friend.
Highlight color green Read1 primer site: The standard TruSeq Read 1 primer site: ACACTCTTTCCCTACACGACGCTCTTCCGATCT. We are not sure at this point whether the small RNA primer site is compatible with dual-indexes or not.
- The remaining template elements are identical to the Single-index adaptor design above.
- Note that an artifact of this design is that a SINGLE index (I7 index) TS library will read the sequence "TCTTTC..." as the I5 index if it is run in dual-index mode.
Dual-index Nextera adaptor design (we believe these are compatible with Illumina V3 PE HiSeq or V2/V3 MiSeq run)
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Nextera® DNA Sample Preparation Kit (Illumina) 1,2
Nextera® transposase sequences (FC-121-1031, FC-121-1030)
5’ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
(a) Read 1 -->
5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
(d) Read 2 -->
Nextera® Index Kit - PCR primers (FC-121-1012, FC-121-1011)
5’ AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC
(c) i5 Index read -->
5’ CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG
<-- i7 Index read (b)
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Nextera codes for entry on sample sheet:
i5 bases in adapter | Nextera DNA i5 index name | Nextera XT i5 index name | Nextera Enrichment i5 index name | HiSeq 2500 and MiSeq i5 bases for entry on sample sheet | NextSeq and HiSeq 4000 i5 bases for entry on sample sheet |
|---|---|---|---|---|---|
TAGATCGC | N501 | S501 | E501 | TAGATCGC | GCGATCTA |
CTCTCTAT | N502 | S502 | E502 | CTCTCTAT | ATAGAGAG |
TATCCTCT | N503 | S503 | E503 | TATCCTCT | AGAGGATA |
AGAGTAGA | N504 | S504 | E504 | AGAGTAGA | TCTACTCT |
GTAAGGAG | N505 | S505 | E505 | GTAAGGAG | CTCCTTAC |
ACTGCATA | N506 | S506 | E506 | ACTGCATA | TATGCAGT |
AAGGAGTA | N507 | S507 | E507 | AAGGAGTA | TACTCCTT |
CTAAGCCT | N508 | S508 | E508 | CTAAGCCT | AGGCTTAG |
i7 bases in adapter | Nextera DNA i7 index name | Nextera XT i7 index name | Nextera Enrichment i7 index name | i7 bases for entry on sample sheet (HiSeq, MiSeq, or NextSeq) | |
TCGCCTTA | N701 | N701 | N701 | TAAGGCGA | |
CTAGTACG | N702 | N702 | N702 | CGTACTAG | |
TTCTGCCT | N703 | N703 | N703 | AGGCAGAA | |
GCTCAGGA | N704 | N704 | N704 | TCCTGAGC | |
AGGAGTCC | N705 | N705 | N705 | GGACTCCT | |
CATGCCTA | N706 | N706 | N706 | TAGGCATG | |
GTAGAGAG | N707 | N707 | N707 | CTCTCTAC | |
CCTCTCTG | N708 | N708 | N708 | CAGAGAGG | |
AGCGTAGC | N709 | N709 | N709 | GCTACGCT | |
CAGCCTCG | N710 | N710 | N710 | CGAGGCTG | |
TGCCTCTT | N711 | N711 | N711 | AAGAGGCA | |
TCCTCTAC | N712 | N712 | N712 | GTAGAGGA | |
Barcodes (also known as Indexes)
NOTE: Illumina barcodes (indexes) have varied significantly over time NOT ONLY in their sequence but also in WHERE they are placed in the sequencing construct.
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The GSAF uses the following names for the following barcodes. Note that these sequences are shown 5'-3' when the P5 sequence is on the left. In other words, here is the first barcode shown in the context of the full 3'-end adaptor construct:
GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG
Sequence | TruSeq name | NEXTFlex number |
|---|---|---|
ATCACG | TSBC01 | NFBC07 |
CGATGT | TSBC02 | NFBC01 |
TTAGGC | TSBC03 | NFBC08 |
TGACCA | TSBC04 | NFBC02 |
ACAGTG | TSBC05 | NFBC03 |
GCCAAT | TSBC06 | NFBC04 |
CAGATC | TSBC07 | NFBC05 |
ACTTGA | TSBC08 | NFBC09 |
GATCAG | TSBC09 | NFBC10 |
TAGCTT | TSBC10 | NFBC11 |
GGCTAC | TSBC11 | NFBC12 |
CTTGTA | TSBC12 | NFBC06 |
AGTCAA | TSBC13 | NFBC13 |
AGTTCC | TSBC14 | NFBC14 |
ATGTCA | TSBC15 | NFBC15 |
CCGTCC | TSBC16 | NFBC16 |
GTAGAG | TSBC17 | NFBC17 |
GTCCGC | TSBC18 | NFBC18 |
GTGAAA | TSBC19 | NFBC19 |
GTGGCC | TSBC20 | NFBC20 |
GTTTCG | TSBC21 | NFBC21 |
CGTACG | TSBC22 | NFBC22 |
GAGTGG | TSBC23 | NFBC23 |
GGTAGC | TSBC24 | NFBC24 |
ACTGAT | TSBC25 | NFBC25 |
ATGAGC | TSBC26 | NFBC26 |
ATTCCT | TSBC27 | NFBC27 |
CAAAAG | TSBC28 | NFBC28 |
CAACTA | TSBC29 | NFBC29 |
CACCGG | TSBC30 | NFBC30 |
CACGAT | TSBC31 | NFBC31 |
CACTCA | TSBC32 | NFBC32 |
CAGGCG | TSBC33 | NFBC33 |
CATGGC | TSBC34 | NFBC34 |
CATTTT | TSBC35 | NFBC35 |
CCAACA | TSBC36 | NFBC36 |
CGGAAT | TSBC37 | NFBC37 |
CTAGCT | TSBC38 | NFBC38 |
CTATAC | TSBC39 | NFBC39 |
CTCAGA | TSBC40 | NFBC40 |
| GTGATC | NEB40 | |
GACGAC | TSBC41 | N/A |
GCGCTA | N/A | NFBC41 |
TAATCG | TSBC42 | NFBC42 |
TACAGC | TSBC43 | NFBC43 |
TATAAT | TSBC44 | NFBC44 |
TCATTC | TSBC45 | NFBC45 |
TCCCGA | TSBC46 | NFBC46 |
TCGAAG | TSBC47 | NFBC47 |
TCGGCA | TSBC48 | NFBC48 |
NOTE that TSBC41 is hamming distance 2 away from both TSBC31 and TSBC11; all others are hamming distance >=3.
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After exhaustive searching of all 4096 6-mers, the following table is all remaining 6 bp barcodes that have hamming distance of at least 3 from each other and the table above of 49 barcodes (NOTE: these have NOT been tested on the sequencer as of 2/7/12):
Sequence | GSAF name |
|---|
AAACAC | UTBC50 |
TGAAGG | UTBC51 |
AACATA | UTBC52 |
CGCGTC | UTBC53 |
GATACA | UTBC54 |
GGTGTG | UTBC55 |
TAAGAA | UTBC56 |
AGCGAG | UTBC57 |
CGGTTA | UTBC58 |
AGCTTT | UTBC59 |
TGGTCT | UTBC60 |
TATCCC | UTBC61 |
TGTCGT | UTBC62 |
CCCCAC | UTBC63 |
ATACGA | UTBC64 |
CCCTTG | UTBC65 |
ACCGGC | UTBC66 |
TTACTG | UTBC67 |
GGAACT | UTBC68 |
GTTATT | UTBC69 |
AAAAGT | UTBC70 |
AAGGGA | UTBC71 |
AAGTAT | UTBC72 |
ACATCT | UTBC73 |
ACGATT | UTBC74 |
ACGCCG | UTBC75 |
ACTCTC | UTBC76 |
AGAATC | UTBC77 |
ATTGGG | UTBC78 |
CCGCGT | UTBC79 |
CGCCCT | UTBC80 |
CTGCAG | UTBC81 |
GAAGTT | UTBC82 |
GCACCC | UTBC83 |
GCAGGA | UTBC84 |
GCCGCG | UTBC85 |
GGCGGT | UTBC86 |
GTATTA | UTBC87 |
TACGTG | UTBC88 |
TCACAT | UTBC89 |
TCTATA | UTBC90 |
TGCAAA | UTBC91 |
TGGCAC | UTBC92 |
TGTTAG | UTBC93 |
TTCTAT | UTBC94 |
| GGTACG | UTBC95 |
Excruciating details - USE WITH CAUTION - RNA PCR primers are NOT current as of Dec. 2011
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Oligonucleotide sequences for TruSeqTM RNA and DNA Sample Prep Kits1
TruSeq Universal Adapter
5’ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
TruSeq Adapters
barcode: ATCACG
TruSeq Adapter, Index 1
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 2
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 3
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 4
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 5
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 6
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 7
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 8
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 9
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 10
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 11
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG
TruSeq Adapter, Index 12
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG
5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC Mux index read seq primer
AGATCGGAAGAGCACACGTCTG - my 3' adaptor
my RT primer:
5' TCAGACGTGTGCTCTTCCGATCT 3'
TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG Mux read 2 seq primer (reversed)
CTTGAGGTCAGTGGAACATTAGAGCATACGGCAGAAGACGAAC Index 12 PCR primer (reversed)
So order RC of TruSeq Adaptor Indexes as PCR primers
Oligonucleotide sequences for TruSeq Small RNA Sample Prep Kits
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Example (all Illumina sequences unless noted):
RNA PCR Primer (RP1), part # 15005505
5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA
AATGATACGGCGACCACCGA CAGGTTCAGAGTTCTACAGTCCGA - this is the NEB SR Primer R1 - pads added by SPHS
5' RNA adaptor GUUCAGAGUUCUACAGUCCGACGAUC (NEB kit calls this the SR Adaptor 1)
RNA PCR Primer, Index 1 (RPI1) code ATCACG in DNA, RC (CGTGAT)
5' CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA 3' - RNA pcr primer
CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC (RC of DNA Index 1 for comparison)
3' RNA adaptor 3' CCTTGGCACCCGAGAATTCCA - 5'
*****************************
Differences with NEB Small RNA kit (RP1/SR Primer R1/SR Adaptor 1/5' RNA adaptor are all the same):
5’ GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG (TruSeq Adaptor Ind 12)
NEB 3' adaptor (3' SR Adaptor 1) 5' ATCGTATGCCGTCTTCTGCTTG 3'
3' AGCATACGGCAGAAGACGAAC 5' (reverse of NEB SR Primer F1)
3' CTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTGCCGATGTAGAGCATACGGCAGAAGACGAAC 5' (rev. of Ind. 12 adaptor)
CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTC (TruSeq Adaptor Ind 12 PCR primer)
5' CAAGCAGAAGACGGCATACGA 3' (NEB SR Primer F1)
5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT (Illumina genomic PCR primer)
3' CAAGCAGAAGACGGCATACGAT 5' - NEB 3' SR Adaptor 1 (RC)
5' CAAGCAGAAGACGGCATACGA 3' - NEB RT Primer 1
For reference:
>TruSeqAdapterIndex11_RC
CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC
>TruSeqAdapterIndex12_RC
CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC
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RNA 5’ Adapter (RA5), part # 15013205
5’ GUUCAGAGUUCUACAGUCCGACGAUC
RNA 3’ Adapter (RA3), part # 15013207
5’ TGGAATTCTCGGGTGCCAAGG
3' ACCTTAAGAGCCCACGGTTCCG 5' (RT primer, reversed)
RNA RT Primer (RTP), part # 15013981
5’ GCCTTGGCACCCGAGAATTCCA
Multiplexing Index Read Sequencing Primer
5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
Multiplexing Read 2 Sequencing Primer
5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
RNA PCR Primer (RP1), part # 15005505
5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA
RNA PCR Primer, Index 1 (RPI1)
CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 2 (RPI2)
CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 3 (RPI3)
CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 4 (RPI4)
CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 5 (RPI5)
CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 6 (RPI6)
CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 7 (RPI7)
CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 8 (RPI8)
CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 9 (RPI9)
CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 10 (RPI10)
CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 11 (RPI11)
CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 12 (RPI12)
CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 13 (RPI13)
CAAGCAGAAGACGGCATACGAGATTTGACTGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 14 (RPI14)
CAAGCAGAAGACGGCATACGAGATGGAACTGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 15 (RPI15)
CAAGCAGAAGACGGCATACGAGATTGACATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 16 (RPI16)
CAAGCAGAAGACGGCATACGAGATGGACGGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 17 (RPI17)
CAAGCAGAAGACGGCATACGAGATCTCTACGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 18 (RPI18)
CAAGCAGAAGACGGCATACGAGATGCGGACGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 19 (RPI19)
CAAGCAGAAGACGGCATACGAGATTTTCACGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 20 (RPI20)
CAAGCAGAAGACGGCATACGAGATGGCCACGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 21 (RPI21)
CAAGCAGAAGACGGCATACGAGATCGAAACGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 22 (RPI22)
CAAGCAGAAGACGGCATACGAGATCGTACGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 23 (RPI23)
CAAGCAGAAGACGGCATACGAGATCCACTCGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 24 (RPI24)
CAAGCAGAAGACGGCATACGAGATGCTACCGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 25 (RPI25)
CAAGCAGAAGACGGCATACGAGATATCAGTGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 26 (RPI26)
CAAGCAGAAGACGGCATACGAGATGCTCATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 27 (RPI27)
CAAGCAGAAGACGGCATACGAGATAGGAATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 28 (RPI28)
CAAGCAGAAGACGGCATACGAGATCTTTTGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 29 (RPI29)
CAAGCAGAAGACGGCATACGAGATTAGTTGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 30 (RPI30)
CAAGCAGAAGACGGCATACGAGATCCGGTGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 31 (RPI31)
CAAGCAGAAGACGGCATACGAGATATCGTGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 32 (RPI32)
CAAGCAGAAGACGGCATACGAGATTGAGTGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 33 (RPI33)
CAAGCAGAAGACGGCATACGAGATCGCCTGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 34 (RPI34)
CAAGCAGAAGACGGCATACGAGATGCCATGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 35 (RPI35)
CAAGCAGAAGACGGCATACGAGATAAAATGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 36 (RPI36)
CAAGCAGAAGACGGCATACGAGATTGTTGGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 37 (RPI37)
CAAGCAGAAGACGGCATACGAGATATTCCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 38 (RPI38)
CAAGCAGAAGACGGCATACGAGATAGCTAGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 39 (RPI39)
CAAGCAGAAGACGGCATACGAGATGTATAGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 40 (RPI40)
CAAGCAGAAGACGGCATACGAGATTCTGAGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 41 (RPI41)
CAAGCAGAAGACGGCATACGAGATGTCGTCGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 42 (RPI42)
CAAGCAGAAGACGGCATACGAGATCGATTAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 43 (RPI43)
CAAGCAGAAGACGGCATACGAGATGCTGTAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 44 (RPI44)
CAAGCAGAAGACGGCATACGAGATATTATAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 45 (RPI45)
CAAGCAGAAGACGGCATACGAGATGAATGAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 46 (RPI46)
CAAGCAGAAGACGGCATACGAGATTCGGGAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 47 (RPI47)
CAAGCAGAAGACGGCATACGAGATCTTCGAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
RNA PCR Primer, Index 48 (RPI48)
CAAGCAGAAGACGGCATACGAGATTGCCGAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA
Oligonucleotide sequences for Genomic DNA
Adapters
5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
PCR Primers
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT
Genomic DNA Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Paired End DNA oligonucleotide sequences
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
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
CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTC
PCR Primer, Index 2
CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTC
PCR Primer, Index 3
CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTC
PCR Primer, Index 4
CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTC
PCR Primer, Index 5
CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTC
PCR Primer, Index 6
CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTC
PCR Primer, Index 7
CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTC
PCR Primer, Index 8
CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTC
PCR Primer, Index 9
CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTC
PCR Primer, Index 10
CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTC
PCR Primer, Index 11
CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTC
PCR Primer, Index 12
CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTC
Oligonucleotide sequences © 2007-2012 Illumina, Inc. All rights reserved.
Derivative works created by Illumina customers are authorized for use with Illumina
instruments and products only. All other uses are strictly prohibited.
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Nextera adaptor style: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGT-3' ||||||||||||||||||| 3'-CAGAGCACCCGAGCCTCTACACATATTCTCTGTC-5' TruSeq truncated adaptor: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' ||||||||||||| 3'-CACTGACCTCAAGTCTGCACACGAGAAGGCTAGA-5' Nextera adaptor style, with primers overlaid: First cycle: 3'-GGCTCGGGTGCTCTG CAAAGC TAGAGCATACGGCAGAAGACGAAC-5' 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGT-3' <UNKNOWN> 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' ||||||||||||||||||| ||||||||||||||||||| 3'-CAGAGCACCCGAGCCTCTACACATATTCTCTGTC-5' <UNKNOWN> 3'-TGACAGAGAATATGTGTAGACTGCGACGGCTGCT-5' Second and subsequent cycles: AATGATACGGCGACCACCGAGATCTACAC ATCACG TCGTCGGCAGCGTC 3'-AGCAGCCGTCGCAGTCTACACATATTCTCTGTCA <UNKNOWN> 3'-TGACAGAGAATATGTGTAGACTGCGACGGCTGCT-5' |
Cautions, common mistakes, and lessons learned from failure
- Assembling the P7 side adaptor or primer wrong - the key thing to note is that the "cannonical designs" are shown 5' to 3' across the entire finished sequencing construct. So if you're designing a reverse primer for the P7 side you have to use the reverse complement of ALL 3 DESIGN ELEMENTS (flow cell binding site, barcode, and sequencing primer site) and make sure they're in the right order.
- Incorrect P5 dual-index design - the "ACAC" motif in the single index design MUST be repeated on both sides of an index within P5 - see the "dual index" designs specifically.
- Reverse complement barcode sequences in either P5 or P7 side indexes, especially from amplicons - the fact that the Illumina sequencers read i5 differently is a pain - pay attention to that when submitting barcode sequences that will wind up in a sample sheet. And remember that the i7 index is read "forward, top strand" of the canonical design, which is reverse complement of the sequence that appears in a reverse primer used when creating a library.