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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.
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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)
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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 |
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 |
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|---|---|---|
AAACAC | UTBC50 |
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TGAAGG | UTBC51 |
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AACATA | UTBC52 |
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CGCGTC | UTBC53 |
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GATACA | UTBC54 |
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GGTGTG | UTBC55 |
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TAAGAA | UTBC56 |
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AGCGAG | UTBC57 |
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CGGTTA | UTBC58 |
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AGCTTT | UTBC59 |
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TGGTCT | UTBC60 |
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TATCCC | UTBC61 |
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TGTCGT | UTBC62 |
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CCCCAC | UTBC63 |
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ATACGA | UTBC64 |
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CCCTTG | UTBC65 |
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ACCGGC | UTBC66 |
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TTACTG | UTBC67 |
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GGAACT | UTBC68 |
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GTTATT | UTBC69 |
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AAAAGT | UTBC70 |
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AAGGGA | UTBC71 |
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AAGTAT | UTBC72 |
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ACATCT | UTBC73 |
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ACGATT | UTBC74 |
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ACGCCG | UTBC75 |
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ACTCTC | UTBC76 |
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AGAATC | UTBC77 |
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ATTGGG | UTBC78 |
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CCGCGT | UTBC79 |
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CGCCCT | UTBC80 |
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CTGCAG | UTBC81 |
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GAAGTT | UTBC82 |
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GCACCC | UTBC83 |
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GCAGGA | UTBC84 |
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GCCGCG | UTBC85 |
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GGCGGT | UTBC86 |
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GTATTA | UTBC87 |
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TACGTG | UTBC88 |
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TCACAT | UTBC89 |
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TCTATA | UTBC90 |
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TGCAAA | UTBC91 |
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TGGCAC | UTBC92 |
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TGTTAG | UTBC93 |
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TTCTAT | UTBC94 |
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Excruciating details - USE WITH CAUTION - RNA PCR primers are NOT current as of Dec. 2011
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