Universal Sequencing TELL-Seq Library
Specifications
- Product Name: TELL-SeqTM Library Sequencing Kit
- Manufacturer: Universal Sequencing Technology Corporation
- Compatibility: All Illumina sequencing systems except iSeq 100
- Intended Use: Research Use Only, not for diagnostic procedures
- Revision History: Version 7.0, August 2024
Product Usage Instructions:
Introduction
A TELL-SeqTM library requires custom sequencing primers for any Illumina sequencing systems and contains an 18-base index 1 sequence used as the molecular barcode for linked reads, which must be sequenced completely.
Kit Contents
| Component Name | Cap Color | Concentration | Volume (µL) |
|---|---|---|---|
| Read 1 Primer | CAP Black | 100M | 50 |
| Read 2 Primer | CAP White | 100M | 50 |
| Index 1 Primer | CAP Red | 100M | 50 |
| Index 2 Primer | CAP Yellow | 100M | 50 |
TELL-SeqTM Library Structure and Sequencing Scheme
- Index 1 (I7 index) contains 18-base TELL Bead sequences that must be sequenced completely.
- Index 2 (I5 index) contains sample index primer sequences used in library amplification.
- Paired end sequencing is preferred with a minimum read length requirement of 2×96 and a maximum read length requirement of 2×150.
- Libraries can be pooled together for sequencing when different multiplex primers are used in the library amplification step.
Custom Sequencing Primers
Custom sequencing primers are required to sequence TELL-SeqTM libraries and are provided in the TELL-SeqTM Illumina Sequencing Primer Kit.
Custom Index 2 Primer
A custom Index 2 primer is needed when multiple TELL-SeqTM libraries with different multiplex primers are pooled for sequencing and when a sequencer requires an i5 index sequencing primer. Currently, only for MiSeq, a custom Index 2 Primer is not required.
Frequently Asked Questions (FAQ)
- Q: Can the TELL-SeqTM kit be used for diagnostic procedures?
A: No, the TELL-SeqTM kit is for Research Use Only and should not be used for diagnostic procedures. - Q: What is the minimum and maximum read length requirement for paired end sequencing?
A: The minimum read length requirement is 2×96 and the maximum read length requirement is 2×150 for paired end sequencing.
TELL-Seq™ Library Sequencing User Guide
For all Illumina@ sequencing systems except iSeq 100
For Research Use Only. Not for use in diagnostic procedures.
Document # 100018 v7.0
August 2024
This document is proprietary to Universal Sequencing Technology Corporation and is intended solely for the use of its customer in connection with the use of the products described herein and for no other purposes.
The instructions in this document must be followed precisely by properly trained personnel to ensure the proper and safe use of the TELL-Seq™ kit.
UNIVERSAL SEQUENCING TECHNOLOGY DOES NOT ASSUME ANY LIABILITY OCCURING AFTER INCORRECT USE OF THE TELL-SEQ™ KIT.
©2022 Universal Sequencing Technology Corporation. All rights reserved.
TELL-Seq™ is a trademark of Universal Sequencing Technology Corporation. All other names, logos and other trademarks are the property of their respective owners.
Revision History
| Document # | Version | DCR Reference and Comment |
| C01K002 Rev. A | January 2020 | Initial Release |
| C01K002 Rev. B | March 2020 | Add Appendix |
| 100018-USG | 3.0 | Add guide for NovaSeq v1.5 reagent |
| 100018-USG | 4.0 | Add detailed guidance for latest NovaSeq
reagents |
| 100018-USG | 5.0 | Add guide for NextSeq 1000/2000 system |
| 100018-USG | 6.0 | Add 96 Multiplex primers (C-series) information |
| 100018-USG | 7.0 | Add 384 Multiplex primers (C,D,E,F-series)
information. Update Index 2 Primer information |
Introduction
This protocol explains how to run any indexed paired-end TELL-Seq™ libraries on an Illumina® sequencing system.
A TELL-Seq™ library† requires custom sequencing primers for any Illumina sequencing systems and contains an 18-base index 1 sequence used as the molecular barcode for linked reads, which must be sequenced completely.
Kit Contents
TELL-Seq™ Illumina® Sequencing Primer Kit (PN 100004)
TELL-Seq™ Illumina® Sequencing Primer Kit, HT (PN 100013)
TELL-Seq™ Library Structure and Sequencing Scheme
Index 1 (i.e., I7 index) contains 18-base TELL Bead sequences, which MUST be sequenced completely. Index 2 (i.e., I5 index) contains 8-base (T-series) or 10-base (C-series) sample index primer sequences used in library amplification. Paired end sequencing is preferred. Minimal read length requirement is 2×96; Maximum read length requirement is 2×150.
Example of Illumina® Sequencing % Base by Cycle Chart
Illumina® Sequencing Guideline
- Dilute TELL-Seq™ library according to Illumina® sequencing platform specific concentration and volume.
- Libraries may be pooled together for sequencing when different multiplex primers are used in the library amplification step.
- Custom sequencing primers are required to sequence TELL-Seq™ libraries and provided in the TELL-Seq™ Illumina Sequencing Primer Kit.
- These custom sequencing primers can be loaded into the specified wells for custom primers. Alternatively, they can also be loaded into corresponding standard Illumina® sequencing primer wells when an Illumina® PhiX control library is spiked in a sequencing run.
- Custom Index 2 primer is only needed when multiple TELL-Seq™ libraries with different multiplex primers are pooled for sequencing and when a sequencer requires an i5 index sequencing primer. Currently only for MiSeq, custom Index 2 Primer is not required. Also for non supported systems like HiSeq 2000/2500 and NovaSeq v1 reagents, custom Index 2 primer is not required.
- The minimum number of sequencing runs can be performed using the amount of sequencing primers provided are varied based on the sequencing system.
| Sequencing System | Is custom Index 2 Primer required? |
| NovaSeq X/X Plus | Yes |
| NovaSeq 6000 | Yes |
| HiSeq 3000/4000 | Yes |
| NextSeq | Yes |
| MiSeq | No |
| MiniSeq | Yes |
Example Sample Sheet for MiSeq System
Below is an example sample sheet for a 2×146 PE run with 18-cycle index 1 (i.e., I7 index) and 8-cycle or 10-base (C-series) index 2 (i.e., I5 index) sequencing using custom sequencing primers for Read 1, Index 1 and Read 2 which will be loaded into custom sequencing primer wells. Because MiSeq uses a P5 primer on the flow cell surface as the sequencing primer for Index 2 reads, TELL-Seq™ custom Index 2 primer is not required for a MiSeq system. The demultiplexing of any pooled libraries will base on the sample index (i.e., index 2). It will be processed by TELL-read analysis pipeline separately after the sequencing run completes.
Illumina® Sequencing Read Length Recommendation
- Paired end sequencing is recommended.
- TELL-Seq™ library Index 1 is 18-base, Index 2 is 8-base (T-series) or 10-base (C-series). There are total 26-base (T-series) or 28-base (C-series) for both indexes compared to total 16-base for standard Illumina dual index.
- The extra 10-cycle required for sequencing TELL-Seq™ library index need to be deducted from read 1 and read 2 sequencing cycles evenly. Since Illumina sequencing reagent guarantee 2 extra cycles, 4-cycle for read 1 and 4-cycle for read 2 need to be deducted respectively.
- Recommended sequencing length are 2×96 PE to 2×146 PE for dual index run; 2×100 PE to 2×150 PE for a single sample run without need for Index 2 read.
Sequencing Depth Consideration
- Adequate sequencing depth is required to get enough TELL Bead coverage. The more TELL Beads used in library amplification to generate a TELL-Seq™ library, the more sequencing reads will be required to get the desired sequencing depth. However, the fewer TELL Beads used for library amplification, the lower the library complexity will be, which may lead to a higher duplication rate of sequencing reads. The balance between TELL Beads used and TELL-Seq™ library complexity required may depend on the genome size and application.
- For de novo assembly application, at least 60x genome coverage of the sample is recommended in general. However, lower sequencing coverage may also be enough depending on the amount of TELL Beads used for library amplification and TELL-Seq™ library complexity.
- For human phasing, at least 500 million cluster reads per sample which is ~40x depth is recommended.
- For targeted sequencing, minimum 100x coverage is recommended.
Library Multiplex Primer Index Sequences (i.e., Index 2 Read): T-series (8-base)
| Library Multiplex
Primer (T-series) |
For Sample Sheet MiSeq | For Sample Sheet NovaSeq,
NovaSeq X, NextSeq, MiniSeq |
| T501 | TGAACCTT | AAGGTTCA |
| T502 | TGCTAAGT | ACTTAGCA |
| T503 | TGTTCTCT | AGAGAACA |
| T504 | TAAGACAC | GTGTCTTA |
| T505 | CTAATCGA | TCGATTAG |
| T506 | CTAGAACA | TGTTCTAG |
| T507 | TAAGTTCC | GGAACTTA |
| T508 | TAGACCTA | TAGGTCTA |
| T509 | CATCCGAA | TTCGGATG |
| T510 | TTATGAGT | ACTCATAA |
| T511 | AGAGGCGC | GCGCCTCT |
| T512 | TAGCCGCG | CGCGGCTA |
| T513 | ACGAATAA | TTATTCGT |
| T514 | TTCGTAGG | CCTACGAA |
| T515 | GATCTGCT | AGCAGATC |
| T516 | CGCTCCGC | GCGGAGCG |
| T517 | AGGCTATA | TATAGCCT |
| T518 | GCCTCTAT | ATAGAGGC |
| T519 | AGGATAGG | CCTATCCT |
| T520 | TCAGAGCC | GGCTCTGA |
| T521 | CTTCGCCT | AGGCGAAG |
| T522 | TAAGATTA | TAATCTTA |
| T523 | AGTAAGTA | TACTTACT |
| T524 | GACTTCCT | AGGAAGTC |
Library Multiplex Primer Index Sequences (i.e. Index 2 Read): C,D,E,F-series (10-base)
| Library Multiplex Primer (C-series) | For Sample Sheet MiSeq | For Sample Sheet NovaSeq, NovaSeq X, NextSeq, MiniSeq |
| C501 | ACGTACGTAC | GTACGTACGT |
| C502 | CATGCATGCA | TGCATGCATG |
| C503 | GTACGTACGT | ACGTACGTAC |
| C504 | TGCATGCATG | CATGCATGCA |
| C505 | ATGCTGATCA | TGATCAGCAT |
| C506 | CACAGCTGTG | CACAGCTGTG |
| C507 | GCTGATCAGC | GCTGATCAGC |
| C508 | TGATCAGCAT | ATGCTGATCA |
| C509 | ATTCAATACT | AGTATTGAAT |
| C510 | CTAGCGCTAG | CTAGCGCTAG |
| C511 | GCTAGTAGTA | TACTACTAGC |
| C512 | TCCAATCAAG | CTTGATTGGA |
| C513 | AATATTGCTG | CAGCAATATT |
| C514 | CGTCGTTACG | CGTAACGACG |
| C515 | GATTGATTCC | GGAATCAATC |
| C516 | TCTAACAATG | CATTGTTAGA |
| C517 | AGAATTGTCA | TGACAATTCT |
| C518 | CTCAGCAATT | AATTGCTGAG |
| C519 | GGTCCTTGTC | GACAAGGACC |
| C520 | AGGCCTGACA | TGTCAGGCCT |
| C521 | CTCCTAGTGG | CCACTAGGAG |
| C522 | GGTTACAGCT | AGCTGTAACC |
| C523 | CTGATTGGCG | CGCCAATCAG |
| C524 | ATTGGTTAGA | TCTAACCAAT |
| C525 | CCATTCAACT | AGTTGAATGG |
| C526 | CAGTATTGAC | GTCAATACTG |
| C527 | GAGTCCTCAA | TTGAGGACTC |
| C528 | AGCTACTACT | AGTAGTAGCT |
| C529 | TAGCTAGCGC | GCGCTAGCTA |
| C530 | GATGCAACAC | GTGTTGCATC |
| C531 | CCTCAGTACA | TGTACTGAGG |
| C532 | CGGTAATTCA | TGAATTACCG |
| C533 | CGCAATGGCT | AGCCATTGCG |
| C534 | GTACGTTGAA | TTCAACGTAC |
| C535 | TTGATCAGTA | TACTGATCAA |
| C536 | GGCCTAACAA | TTGTTAGGCC |
| C537 | GTTGTTGGAA | TTCCAACAAC |
| C538 | TACGTTGGAC | GTCCAACGTA |
| C539 | ACACCATGCA | TGCATGGTGT |
| C540 | GCAATAGTAC | GTACTATTGC |
| C541 | ACGCAGCCAG | CTGGCTGCGT |
| C542 | CGAGTTGACG | CGTCAACTCG |
| C543 | CGTGGCTGAA | TTCAGCCACG |
| C544 | TCTCAAGGAC | GTCCTTGAGA |
| C545 | CCTAGGCACT | AGTGCCTAGG |
| C546 | CTGCGGTAAT | ATTACCGCAG |
| C547 | GGCACTACCA | TGGTAGTGCC |
| C548 | GCTCAATCAA | TTGATTGAGC |
| C549 | AGGCACACAC | GTGTGTGCCT |
| C550 | CCTGGCAAGA | TCTTGCCAGG |
| C551 | TAATTGGTAG | CTACCAATTA |
| C552 | GCCAACAAGT | ACTTGTTGGC |
| C553 | ATGGCTTATA | TATAAGCCAT |
| C554 | GCATGGCCTT | AAGGCCATGC |
| C555 | ACAATACTGG | CCAGTATTGT |
| C556 | GGATTGGACT | AGTCCAATCC |
| C557 | ACTGTACTAT | ATAGTACAGT |
| C558 | CAGCTGTGAG | CTCACAGCTG |
| C559 | CTTGAGGACC | GGTCCTCAAG |
| C560 | GGTACAATAG | CTATTGTACC |
| C561 | CTGACTACTA | TAGTAGTCAG |
| C562 | TCAACCATGG | CCATGGTTGA |
| C563 | ATTATAACCG | CGGTTATAAT |
| C564 | ACTAGTCCTT | AAGGACTAGT |
| C565 | ACTTGGACGT | ACGTCCAAGT |
| C566 | ATGGTTAGGA | TCCTAACCAT |
| C567 | ATGGTACCAA | TTGGTACCAT |
| C568 | GAATTGACTC | GAGTCAATTC |
| C569 | AGCAACCAGG | CCTGGTTGCT |
| C570 | TACTGTGCTG | CAGCACAGTA |
| C571 | CAACAACGTC | GACGTTGTTG |
| C572 | CAGTAGCGCT | AGCGCTACTG |
| C573 | ATTACCAATC | GATTGGTAAT |
| C574 | TAAGGACCGC | GCGGTCCTTA |
| C575 | ACACGTACCG | CGGTACGTGT |
| C576 | CAACGTTGTT | AACAACGTTG |
| C577 | ATTGTGCTGA | TCAGCACAAT |
| C578 | GTACCAACAG | CTGTTGGTAC |
| C579 | TTGTCAAGGA | TCCTTGACAA |
| C580 | CTTGTACGTA | TACGTACAAG |
| C581 | TGCCTTGTAA | TTACAAGGCA |
| C582 | TAGTAGCTTA | TAAGCTACTA |
| C583 | GACCGCAATG | CATTGCGGTC |
| C584 | CTACTAGCTT | AAGCTAGTAG |
| C585 | AGCACACGTT | AACGTGTGCT |
| C586 | TGTTATAAGC | GCTTATAACA |
| C587 | GTTGCCAAGT | ACTTGGCAAC |
| C588 | CTGGCAACCG | CGGTTGCCAG |
| C589 | TTAGGCCTTA | TAAGGCCTAA |
| C590 | CGCAGCACAG | CTGTGCTGCG |
| C591 | CTAGGCACAA | TTGTGCCTAG |
| C592 | TGTTGTACAG | CTGTACAACA |
| C593 | CTAACGTGGC | GCCACGTTAG |
| C594 | GCGTACTGGT | ACCAGTACGC |
| C595 | GGCCTGAATT | AATTCAGGCC |
| C596 | CATGCTCGAG | CTCGAGCATG |
| Library Multiplex Primer (D-series) | For Sample Sheet MiSeq | For Sample Sheet NovaSeq, NovaSeq X, NextSeq, MiniSeq |
| D501 | AGCACTGTAA | TTACAGTGCT |
| D502 | CCGAAGTACT | AGTACTTCGG |
| D503 | GTTACAAGTA | TACTTGTAAC |
| D504 | TGATTGCGTG | CACGCAATCA |
| D505 | ATCTTAGGCA | TGCCTAAGAT |
| D506 | CACGCAAGTT | AACTTGCGTG |
| D507 | GAGCTTAGGA | TCCTAAGCTC |
| D508 | TATTGCTCGA | TCGAGCAATA |
| D509 | AACATCTGAG | CTCAGATGTT |
| D510 | CAACTCAGCA | TGCTGAGTTG |
| D511 | GAACTATATG | CATATAGTTC |
| D512 | TAATAGTCTA | TAGACTATTA |
| D513 | AAGATTGACG | CGTCAATCTT |
| D514 | CACGTAGCCG | CGGCTACGTG |
| D515 | GACTCGATCA | TGATCGAGTC |
| D516 | TAATCCTCGC | GCGAGGATTA |
| D517 | AAGCTCCTGG | CCAGGAGCTT |
| D518 | CAGATTGTTG | CAACAATCTG |
| D519 | GATGCTATTC | GAATAGCATC |
| D520 | TAATCCTCGC | GCGAGGATTA |
| D521 | AAGTATACCT | AGGTATACTT |
| D522 | CAGTAGCCAA | TTGGCTACTG |
| D523 | GCAAGGACTC | GAGTCCTTGC |
| D524 | TACTACTGTA | TACAGTAGTA |
| D525 | AATCCTAGCT | AGCTAGGATT |
| D526 | CATTGGCAAG | CTTGCCAATG |
| D527 | GCCTGAGAAT | ATTCTCAGGC |
| D528 | TACTAGCAAG | CTTGCTAGTA |
| D529 | ACAGCTAGTC | GACTAGCTGT |
| D530 | CCAACCAGTA | TACTGGTTGG |
| D531 | GCGCTCAATT | AATTGAGCGC |
| D532 | TAGCGCGGAC | GTCCGCGCTA |
| D533 | ACCTATAGTT | AACTATAGGT |
| D534 | CCACGTCAGG | CCTGACGTGG |
| D535 | GCTACGCAAT | ATTGCGTAGC |
| D536 | TAGTTGCTGC | GCAGCAACTA |
| D537 | ACGGTACAAC | GTTGTACCGT |
| D538 | CCGAACTTCG | CGAAGTTCGG |
| D539 | GCTCCTAATT | AATTAGGAGC |
| D540 | TATCCTCGAT | ATCGAGGATA |
| D541 | ACTCGATTCT | AGAATCGAGT |
| D542 | CCTGGTAATT | AATTACCAGG |
| D543 | GCTTAGTTGG | CCAACTAAGC |
| D544 | TCAGCAAGGA | TCCTTGCTGA |
| D545 | ACTGGTAGCA | TGCTACCAGT |
| D546 | CGATCAACCT | AGGTTGATCG |
| D547 | GGAAGTAGCA | TGCTACTTCC |
| D548 | TCAGCTGCGG | CCGCAGCTGA |
| D549 | ACTGGTAGCA | TGCTACCAGT |
| D550 | CGATCAACCT | AGGTTGATCG |
| D551 | GGCAAGATGA | TCATCTTGCC |
| D552 | TCCGCCAATG | CATTGGCGGA |
| D553 | AGAATCTTAT | ATAAGATTCT |
| D554 | CGCTAGCAAC | GTTGCTAGCG |
| D555 | GGCCTTATAT | ATATAAGGCC |
| D556 | TCGAATACTA | TAGTATTCGA |
| D557 | AGCAATGGTA | TACCATTGCT |
| D558 | CGGTCTGATC | GATCAGACCG |
| D559 | GGTCCGCCAT | ATGGCGGACC |
| D560 | TCGAATACTA | TAGTATTCGA |
| D561 | AGCACTGTAA | TTACAGTGCT |
| D562 | CGTACTACCG | CGGTAGTACG |
| D563 | GGTTCATCGG | CCGATGAACC |
| D564 | TCGGAGCTGC | GCAGCTCCGA |
| D565 | AGGACGTTAG | CTAACGTCCT |
| D566 | CGTTGACAAT | ATTGTCAACG |
| D567 | GTACTGACAC | GTGTCAGTAC |
| D568 | TCTAAGATAG | CTATCTTAGA |
| D569 | AGTGCTCAAC | GTTGAGCACT |
| D570 | CTAAGAACCT | AGGTTCTTAG |
| D571 | GTATAATTAC | GTAATTATAC |
| D572 | TGATTGCGTG | CACGCAATCA |
| D573 | ATATTCGACG | CGTCGAATAT |
| D574 | CTATAACGAC | GTCGTTATAG |
| D575 | GTCGTTAGGT | ACCTAACGAC |
| D576 | TGCCACTGAT | ATCAGTGGCA |
| D577 | ATCGGCTAAG | CTTAGCCGAT |
| D578 | CTCAAGTTGC | GCAACTTGAG |
| D579 | GTGTGTTCGA | TCGAACACAC |
| D580 | TGCTATAGTC | GACTATAGCA |
| D581 | ATGAGCTATT | AATAGCTCAT |
| D582 | CTCACAACGA | TCGTTGTGAG |
| D583 | TGCTCGATTG | CAATCGAGCA |
| D584 | ATGTTGGACT | AGTCCAACAT |
| D585 | CTGATGTTAG | CTAACATCAG |
| D586 | TGGTAACCAG | CTGGTTACCA |
| D587 | ATTCGAAGTA | TACTTCGAAT |
| D588 | CTTAGCTCCT | AGGAGCTAAG |
| D589 | TGTAATCGAT | ATCGATTACA |
| D590 | CTTGGACTTC | GAAGTCCAAG |
| D591 | TGTACTTGAA | TTCAAGTACA |
| D592 | TTAAGCTCAG | CTGAGCTTAA |
| D593 | TTACAACCAG | CTGGTTGTAA |
| D594 | TTCTCAGCCA | TGGCTGAGAA |
| D595 | TTGCATGGCT | AGCCATGCAA |
| D596 | TTGGCGGTAC | GTACCGCCAA |
| Library Multiplex Primer (E-series) | For Sample Sheet MiSeq | For Sample Sheet NovaSeq, NovaSeq X, NextSeq, MiniSeq |
| E501 | AGCCACGGTC | GACCGTGGCT |
| E502 | CCGCCTGAGT | ACTCAGGCGG |
| E503 | GTTATATACC | GGTATATAAC |
| E504 | CTTCCTGGCA | TGCCAGGAAG |
| E505 | ATCGAATTGC | GCAATTCGAT |
| E506 | CACGTGTTGT | ACAACACGTG |
| E507 | GAGCTGTGTT | AACACAGCTC |
| E508 | TATTACAGAT | ATCTGTAATA |
| E509 | AACAATGGCG | CGCCATTGTT |
| E510 | CAACTCGCTA | TAGCGAGTTG |
| E511 | GAATTGCTCA | TGAGCAATTC |
| E512 | GTGTGTTCGA | TCGAACACAC |
| E513 | CGATTCGAGC | GCTCGAATCG |
| E514 | GTCAACGTTA | TAACGTTGAC |
| E515 | GACAATCCTA | TAGGATTGTC |
| E516 | TAATCAGACT | AGTCTGATTA |
| E517 | AAGCTTATAT | ATATAAGCTT |
| E518 | CATATTGTAA | TTACAATATG |
| E519 | GATAGGACTT | AAGTCCTATC |
| E520 | ATGCTAGGTT | AACCTAGCAT |
| E521 | GCAAGATTAG | CTAATCTTGC |
| E522 | CAGCTGTATA | TATACAGCTG |
| E523 | GCAAGTCTAA | TTAGACTTGC |
| E524 | TACTAGCAAG | CTTGCTAGTA |
| E525 | AATACTGTTA | TAACAGTATT |
| E526 | CATATCTGAC | GTCAGATATG |
| E527 | GCCACTAAGG | CCTTAGTGGC |
| E528 | CTGGCAATTC | GAATTGCCAG |
| E529 | ACAATAGGCG | CGCCTATTGT |
| E530 | CCATGCTAAG | CTTAGCATGG |
| E531 | GCGAGCGATC | GATCGCTCGC |
| E532 | TAGGTAGTTC | GAACTACCTA |
| E533 | ACCTTGGACC | GGTCCAAGGT |
| E534 | TGACTAATAC | GTATTAGTCA |
| E535 | GCTATATCTG | CAGATATAGC |
| E536 | TCTAGTTCAG | CTGAACTAGA |
| E537 | ACGGTTGAGA | TCTCAACCGT |
| E538 | CTGATTCCGG | CCGGAATCAG |
| E539 | TGGTAACCAG | CTGGTTACCA |
| E540 | GACTCAGACA | TGTCTGAGTC |
| E541 | TTAGCCAATA | TATTGGCTAA |
| E542 | CCTTAAGGCG | CGCCTTAAGG |
| E543 | CGTAGCAATC | GATTGCTACG |
| E544 | CATGGCTAAT | ATTAGCCATG |
| E545 | ACTCGACAAT | ATTGTCGAGT |
| E546 | CGAGTCTAGG | CCTAGACTCG |
| E547 | GGACTCAGCT | AGCTGAGTCC |
| E548 | TCAGCAAGGA | TCCTTGCTGA |
| E549 | CGATTCCTTA | TAAGGAATCG |
| E550 | ATTAGGCAAT | ATTGCCTAAT |
| E551 | TTGTCGAAGG | CCTTCGACAA |
| E552 | TCCAAGTAGC | GCTACTTGGA |
| E553 | AGATAGTCCG | CGGACTATCT |
| E554 | CGCTAACATA | TATGTTAGCG |
| E555 | GGCTAATTGT | ACAATTAGCC |
| E556 | AGGTACGTCA | TGACGTACCT |
| E557 | TTCCAAGTTC | GAACTTGGAA |
| E558 | CGGAGCTCCG | CGGAGCTCCG |
| E559 | GGTACTAATA | TATTAGTACC |
| E560 | TCGAAGGCAA | TTGCCTTCGA |
| E561 | CTCCTTGATG | CATCAAGGAG |
| E562 | GCAATTCCGG | CCGGAATTGC |
| E563 | ACTACGCAGC | GCTGCGTAGT |
| E564 | AGGAATGGCC | GGCCATTCCT |
| E565 | AGGTTAGCAT | ATGCTAACCT |
| E566 | CGTCCTAGAC | GTCTAGGACG |
| E567 | GCGTGGCAAT | ATTGCCACGC |
| E568 | TCTGATCGAC | GTCGATCAGA |
| E569 | AGTTGTCAGG | CCTGACAACT |
| E570 | CTACGCCAAT | ATTGGCGTAG |
| E571 | GTAGTTGCGT | ACGCAACTAC |
| E572 | TGAGCGCTAT | ATAGCGCTCA |
| E573 | ATACCTTAGT | ACTAAGGTAT |
| E574 | GCACACCATT | AATGGTGTGC |
| E575 | GTCTAAGGAG | CTCCTTAGAC |
| E576 | CATTCCGCGG | CCGCGGAATG |
| E577 | GCGTACCTAG | CTAGGTACGC |
| E578 | CTCAATAGCA | TGCTATTGAG |
| E579 | GTGCTAAGCC | GGCTTAGCAC |
| E580 | TGCCACTATC | GATAGTGGCA |
| E581 | GCTGTTGAAT | ATTCAACAGC |
| E582 | CGGCTAATCC | GGATTAGCCG |
| E583 | AACTACCGCA | TGCGGTAGTT |
| E584 | ATGCATCAAG | CTTGATGCAT |
| E585 | CTGACTTGCA | TGCAAGTCAG |
| E586 | TGGTACCTAT | ATAGGTACCA |
| E587 | ATTAGCTAAC | GTTAGCTAAT |
| E588 | TGGTACCTAT | ATAGGTACCA |
| E589 | TGTTGCTACC | GGTAGCAACA |
| E590 | CTTATATAAG | CTTATATAAG |
| E591 | TCAAGTCTGT | ACAGACTTGA |
| E592 | TGAGCGCTAT | ATAGCGCTCA |
| E593 | TTAGGCGTAC | GTACGCCTAA |
| E594 | TTCTCATCAG | CTGATGAGAA |
| E595 | CTAGGTATTG | CAATACCTAG |
| E596 | TTGCTGACTA | TAGTCAGCAA |
| Library Multiplex Primer (F-series) | For Sample Sheet MiSeq | For Sample Sheet NovaSeq, NovaSeq X, Next Seq, MiniSeq |
| F501 | AGCCAGTAGC | GCTACTGGCT |
| F502 | CCGATGAGTT | AACTCATCGG |
| F503 | GTTCAACTTC | GAAGTTGAAC |
| F504 | TTGAGCATCA | TGATGCTCAA |
| F505 | ATCAACAGTG | CACTGTTGAT |
| F506 | CACACGGTAG | CTACCGTGTG |
| F507 | GAGGATTGCT | AGCAATCCTC |
| F508 | TATCTTGGCC | GGCCAAGATA |
| F509 | AACTAGTGGC | GCCACTAGTT |
| F510 | CAATCCTGGT | ACCAGGATTG |
| F511 | GAAGGTTACG | CGTAACCTTC |
| F512 | CTAAGAACCT | AGGTTCTTAG |
| F513 | CACACGGTAG | CTACCGTGTG |
| F514 | AATTAGTCTG | CAGACTAATT |
| F515 | GACCGTGTTA | TAACACGGTC |
| F516 | TAATTCGAGG | CCTCGAATTA |
| F517 | AAGATTGACG | CGTCAATCTT |
| F518 | CAACTCGCTA | TAGCGAGTTG |
| F519 | GATTCGAACT | AGTTCGAATC |
| F520 | GCCTTGAGTT | AACTCAAGGC |
| F521 | CACAGATTAG | CTAATCTGTG |
| F522 | CAGCTAGGCC | GGCCTAGCTG |
| F523 | GCAAGGTATA | TATACCTTGC |
| F524 | TACCTAGGTA | TACCTAGGTA |
| F525 | AATCCTTGAA | TTCAAGGATT |
| F526 | CATCGAACCT | AGGTTCGATG |
| F527 | GCCTGCGTAA | TTACGCAGGC |
| F528 | CATCGAACCT | AGGTTCGATG |
| F529 | ACAGTGCCAT | ATGGCACTGT |
| F530 | CCAACTATTG | CAATAGTTGG |
| F531 | GCGTACCGCA | TGCGGTACGC |
| F532 | TAGGAGCGAT | ATCGCTCCTA |
| F533 | ACCAATTGGT | ACCAATTGGT |
| F534 | AATACTGTTA | TAACAGTATT |
| F535 | GCTGTTGAAT | ATTCAACAGC |
| F536 | TTACAACCAG | CTGGTTGTAA |
| F537 | ACGTCAAGTC | GACTTGACGT |
| F538 | CAATTCAATG | CATTGAATTG |
| F539 | TGAATTCAGC | GCTGAATTCA |
| F540 | ACGTACAGCT | AGCTGTACGT |
| F541 | TGACTACCAT | ATGGTAGTCA |
| F542 | CCTGGTGTGA | TCACACCAGG |
| F543 | GACTAATCGC | GCGATTAGTC |
| F544 | ACGTTAGTGC | GCACTAACGT |
| F545 | ACTCGATTCT | AGAATCGAGT |
| F546 | CGACAAGATT | AATCTTGTCG |
| F547 | GGAATTGGCT | AGCCAATTCC |
| F548 | TCACAGCCGA | TCGGCTGTGA |
| F549 | CTGTACTGTA | TACAGTACAG |
| F550 | TCGCTCGAGG | CCTCGAGCGA |
| F551 | ACCTTAGCAA | TTGCTAAGGT |
| F552 | TCCGGACTAC | GTAGTCCGGA |
| F553 | AGAACTTGCA | TGCAAGTTCT |
| F554 | CGCAGGACTT | AAGTCCTGCG |
| F555 | GGCAAGATGA | TCATCTTGCC |
| F556 | GCCTAGCGCG | CGCGCTAGGC |
| F557 | GACATCGCTA | TAGCGATGTC |
| F558 | CGGCTAATCC | GGATTAGCCG |
| F559 | GGTTGAGCTT | AAGCTCAACC |
| F560 | TCGCTCAGGC | GCCTGAGCGA |
| F561 | GCTGATTATA | TATAATCAGC |
| F562 | GCGCTAGTCC | GGACTAGCGC |
| F563 | TATGTCTGAA | TTCAGACATA |
| F564 | AAGCTTATAT | ATATAAGCTT |
| F565 | AGGAATGGCC | GGCCATTCCT |
| F566 | CGTAGCGCGC | GCGCGCTACG |
| F567 | GCCTGCGTAA | TTACGCAGGC |
| F568 | TCTTAGCGAT | ATCGCTAAGA |
| F569 | AGTTCTAAGA | TCTTAGAACT |
| F570 | CTAGGTATTG | CAATACCTAG |
| F571 | GTAGTACTAC | GTAGTACTAC |
| F572 | TGACAAGTAG | CTACTTGTCA |
| F573 | ATAGTCTGAG | CTCAGACTAT |
| F574 | CTGTGTTACA | TGTAACACAG |
| F575 | GTCTAGCCAC | GTGGCTAGAC |
| F576 | AATCAAGGTT | AACCTTGATT |
| F577 | CATAATTATG | CATAATTATG |
| F578 | CTCGAGCTAT | ATAGCTCGAG |
| F579 | GTGCCTGATG | CATCAGGCAC |
| F580 | TGCTCGATTG | CAATCGAGCA |
| F581 | GATTCGAACT | AGTTCGAATC |
| F582 | TCGTTGAGGA | TCCTCAACGA |
| F583 | AGCCAGGCGT | ACGCCTGGCT |
| F584 | ATGTCAGATT | AATCTGACAT |
| F585 | CTGTATATAC | GTATATACAG |
| F586 | TGGTGTCAAC | GTTGACACCA |
| F587 | ATTGCATGCT | AGCATGCAAT |
| F588 | AGTATTCATA | TATGAATACT |
| F589 | TGTAATTCCG | CGGAATTACA |
| F590 | CTTCCTGGCA | TGCCAGGAAG |
| F591 | TCCGCGCGGA | TCCGCGCGGA |
| F592 | CACTGTTCGG | CCGAACAGTG |
| F593 | TTAGCCAATA | TATTGGCTAA |
| F594 | TTCGAGCTGA | TCAGCTCGAA |
| F595 | GTACCGCGCG | CGCGCGGTAC |
| F596 | TTGAGCATCA | TGATGCTCAA |
Appendix
Spiking TELL-Seq™ Custom Primers into Illumina® Sequencing Primers
TELL-Seq™ libraries require custom sequencing primers for Illumina sequencing platforms. Spiking (or combining) custom TELL-Seq™ Illumina sequencing primers into the standard Illumina sequencing primers is necessary when including PhiX control library or other standard Illumina libraries with TELL- Seq™ libraries in a sequencing run.
Note: TELL-Seq™ Index 1 read has 18-base and need 18-cycle sequencing; Index 2 read has 8-base for T- series primer and 10-base for C-series primer.
Procedure for spiking custom primers into Illumina primers
- The cartridge position, total volumes, and final concentration of custom primers for each platform are provided in the tables below.
- Calculate the volume of the custom primer to add to the Illumina primer cartridge position based on the final concentration of the custom primer in the cartridge.*
- After spiking in the custom primer, adjust the pipette to half of the total volume and gently pipette up and down five times to mix thoroughly.
- For MiSeq, MiniSeq, NextSeq and NovaSeq platforms, do not check the “custom primer” box position in the sample sheet or during the run setup.
* To calculate how much custom primer to spike into the well, use the (C1)*(V1) = (C2)*(V2) equation where:
- C1 = 100μM (when using 100μM high concentration primer stock, the small additional volume to the final volume is negligible).
- V1 = solve for the volume of the custom primer to be spiked in
- C2 = the recommended custom primer final concentration from the chart below V2 = total volume of Illumina primer in the charts below
Example for MiSeq platform
100μM * V1 = 0.5μM * 680μL V1 = 3.4 µl
Important Note: The guidelines below are based on the current primer volumes. To ensure accuracy, measure total primer volumes in the cartridge with a pipette before proceeding with setup.
Each Illumina® sequencer has different final concentration and total volume so refer to document below to ensure that appropriate amount of TELL-Seq™ Illumina® Sequencing Primers are being directly added.
*Note: Index 1 and Index 2 along with Read 1 and Read 2 primers are often pooled together in reagent cartridge well. Please ensure that final concentration custom primer for the well is of each primer (eg. 0.3μM of Index 1 and Index 2 for a total of 0.6μM)
<https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000001542>
Below are
iSeq100
Due to the construction of the iSeq100 cartridge, it is not possible to load and use custom primers.
MiniSeq
NextSeq
MiSeq
* There is no option for a custom Index 2 (i5) primer since the template uses the grafted P5 primer on the surface of the flow cell.
HiSeq 1000/2000 – 1500/2300 
* There is no option for a custom Index 2 (i5) primer since the template uses the grafted P5 primer on the surface of the flow cell.
HiSeq 3000/4000
NovaSeq v1.0 Consumables
| Kit version | Illumina Primer (name) | Cartridge Position | Total Volume (mL) | Custom primer final concentration (µM) |
|
SP 100/200/300/500 cycles |
Read 1 (BP10) | 24 | 4 | 0.3 |
| Index 1 (i7) (BP14)* | 23 | 5 | 0.3 | |
| Read 2 (BP11) | 13 | 2 | 0.3 | |
|
S1 and S2 100/200/300 cycles |
Read 1 (BP10) | 24 | 4 | 0.3 |
| Index 1 (i7) (BP14)* | 23 | 5 | 0.3 | |
| Read 2 (BP11) | 13 | 2 | 0.3 | |
|
S4 200/300 cycles |
Read 1 (BP10) | 24 | 7.3 | 0.3 |
| Index 1 (i7) (BP14)* | 23 | 5 | 0.3 | |
| Read 2 (BP11) | 13 | 3.5 | 0.3 |
* There is no option for a custom Index 2 (i5) primer since the template uses the grafted P5 primer on the surface of the flow cell.
NovaSeq v1.5 Consumables
| Kit version | Illumina Primer (name) | Cartridge Position | Total Volume (mL) | Custom primer final concentration (µM) |
|
SP 100/200/300/500 cycles |
Read 1 (VP10) | 24 | 4 | 0.3 |
| Index 1 (i7) and Index 2 (i5) (VP14) | 23 | 5 | 0.3 | |
| Read 2 (VP11) | 13 | 2 | 0.3 | |
|
S1 and S2 100/200/300 cycles |
Read 1 (VP10) | 24 | 4 | 0.3 |
| Index 1 (i7) and Index 2 (i5) (VP14) | 23 | 5 | 0.3 | |
| Read 2 (VP11) | 13 | 2 | 0.3 | |
|
S4 200/300 cycles |
Read 1 (VP10) | 24 | 7.3 | 0.3 |
| Index 1 (i7) and Index 2 (i5) (VP14) | 23 | 5 | 0.3 | |
| Read 2 (VP11) | 13 | 3.5 | 0.3 |
NovaSeq X/X Plus
Setting Up a TELL-Seq™ Run on a NextSeq™ 1000/2000 System
The NextSeq™ 1000/2000 reagent cartridge has two custom wells (1 and 2, both are empty) to be used when the library requires at least one custom primer. It supports up to two custom primers (pool) for each custom well. It is essential that the Read primer and Index primer are in different wells.
TELL-Seq™ libraries require custom primers for all reads (read 1, read 2, index 1 and index 2). When only TELL-Seq™ libraries are sequenced in a run
- Combine TELL-Seq™ read 1 and read 2 primers: use HT1 to dilute each custom read primer mix to yield 600 µl at 0.3 µM final concentration, i.e., 1.8 µl of 100 µM TELL-Seq™ read 1 primer and 1.8 µl of 100 µM TELL-Seq™ read 2 primer into 597 µl HT1. Load it into custom well 1.
- Combine TELL-Seq™ index 1 and index 2 primers: use HT1 to dilute each custom index primer mix to yield 600 µl at 0.6 µM final concentration, i.e., 3.6 µl of 100 µM TELL-Seq™ index 1 primer and 3.6 µl of 100 µM TELL-Seq™ index 2 primer into 593 µl HT1. Load it into custom well 2.
- Choose the proper custom primer well when setting up the sequencing run as following:
- Read 1: Custom 1
- Index 1: Custom 2
- Index 2: Custom 2
- Read 2: Custom 1
When PhiX libraries are used with TELL-Seq™ libraries for sequencing in a run
- Obtain a NextSeq 1000/2000 Read Primer Kit (Catalog# 20046117)
Combine TELL-Seq™ read 1 and read 2 primers into HP21: Add each custom read primer mix to 600 µl HP21 for a 0.3 µM final concentration, i.e., 1.8 µl of 100 µM TELL-Seq™ read 1 primer and 1.8 µl of 100 µM TELL-Seq™ read 2 primer into 597 µl HP21. Load it into custom well 1.- Combine TELL-Seq™ index 1 and index 2 primers: use HT1 to dilute each custom index primer mix to yield 600 µl at 0.6 µM final concentration, i.e., 3.6 µl of 100 µM TELL-Seq™ index 1 primer and 3.6 µl of 100 µM TELL-Seq™ index 2 primer into 593 µl HT1. Load it into custom well 2.
- Choose proper custom primer well when setting up the sequencing run as following:
- Read 1: Custom 1
- Index 1: Custom 2
- Index 2: Custom 2
- Read 2: Custom 1
When dual index Illumina libraries are sequenced with TELL-Seq™ libraries together in a run
- Obtain a NextSeq 1000/2000 Read & Index Primer Kit (Catalog# 20046115)
- Combine TELL-Seq™ read 1 and read 2 primers into HP21: Add each custom read primer mix to 600 µl HP21 for a 0.3 µM final concentration, i.e., 1.8 µl of 100 µM TELL-Seq™ read 1 primer and 1.8 µl of 100 µM TELL-Seq™ read 2 primer into 597 µl HP21. Load it into custom well 1.
- Combine TELL-Seq™ index 1 and index 2 primers into BP14: Add each custom index primer mix to 600 µl BP14 for a 0.6 µM final concentration, i.e., 3.6 µl of 100 µM TELL-Seq™ index 1 primer and 3.6 µl of 100 µM TELL-Seq™ index 2 primer into 593 µl BP14. Load it into custom well 2.
- Choose proper custom primer well when setting up the sequencing run as following:
- Read 1: Custom 1
- Index 1: Custom 2
- Index 2: Custom 2
- Read 2: Custom 1
Documents / Resources
 |
Universal Sequencing TELL-Seq Library [pdf] User Guide TELL-Seq Library, TELL-Seq, Library |
