SOLiD TM 4 System. Updates to Paired-End Sequencing on the SOLiD 4 System. In this user bulletin. Purpose USER BULLETIN AUGUST 2010

Transcription

1 SOLiD TM 4 System USER BULLETIN AUGUST 2010 SUBJECT: Updates to Paired-End Sequencing on the SOLiD 4 System In this user bulletin Purpose The user bulletin covers: Purpose Ordering information Updated workflow for 50/35 bp paired-end sequencing Protocol changes Install reagent strip(s) on the instrument Create a sequencing (non-multiplex) run record Create a multiplex sequencing run record Start the sequencing run Recommended fill volumes for on-instrument reagents Times for processes Discontinued kits This user bulletin describes new paired-end sequencing kits and a new starter pack for sequencing up to 50 bp in the forward direction and 35 bp in the reverse direction: SOLiD ToP Paired End Sequencing Kit - DNA Frag. Lib., MM50/35 (PN ) SOLiD ToP Paired End Sequencing Kit - RNA Frag. Lib., MM50/35 (PN ) SOLiD ToP Paired End Sequencing Kit - BC Frag. Lib., MM50/35/5 (PN ) SOLiD ToP Paired End Sequencing Kit - BC Frag. Lib., MM50/35/10 (PN ) SOLiD 4 Paired End Starter Pack v2 (PN ) The new kits replace the SOLiD ToP Paired End 50/25 bp and 35/25 bp sequencing kits. The 50/25 and 35/25 bp sequencing kits will be discontinued by July 30, 2010 (see Discontinued kits on page 7). In addition, the new SOLiD 4 Paired End Starter Pack v2 (PN ) replaces the SOLiD 4 Paired End Starter Pack (PN ). Customers can order a SOLiD 4 Paired End Starter Pack v2 with the purchase of a SOLiD 4 System. Purchase of the starter pack is optional and must be ordered and purchased separately.

2 Ordering information Ordering information New SOLiD ToP Paired End 50/35 bp Sequencing Kits Item Components Kit components used in SOLiD ToP Paired End Sequencing Kit DNA Frag. Lib., MM50/35 (PN ) SOLiD ToP Paired End Sequencing Kit RNA Frag. Lib., MM50/35 (PN ) SOLiD Top Paired End Sequencing Kit BC Frag. Lib., MM50/35/5 (PN ) SOLiD ToP Paired End Sequencing Kit BC Frag. Lib., MM50/35/10 (PN ) SOLiD ToP Paired End Seq. Kit Frag. Lib., F5-P2 Tag, MM35 (PN ) SOLiD Top Paired End Seq. Kit BC Frag. Lib., F5-BC Tag, MM35 (PN ) SOLiD ToP Sequencing Kit Frag. Lib., F3 Tag, MM50 SOLiD ToP Paired End Seq. Kit Frag. Lib., F5-P2 Tag, MM35 SOLiD ToP Sequencing Kit Frag. Lib., F3 Tag, MM50 SOLiD ToP Paired End Seq. Kit BC Frag. Lib., F5-BC Tag, MM35 SOLiD ToP Sequencing Kit Frag. Lib., F3 Tag, MM50 SOLiD ToP Paired End Seq. Kit BC Frag. Lib., F5-BC Tag, MM35 SOLiD ToP Sequencing Kit BC Frag. Lib., BC Tag, MM5 Reset Buffer SOLiD ToP Sequencing Kit Frag. Lib., F3 Tag, MM50 SOLiD ToP Paired End Seq. Kit BC Frag. Lib., F5-BC Tag, MM35 SOLiD ToP Sequencing Kit BC Frag. Lib., BC Tag, MM10 Reset Buffer Non-multiplex paired-end sequencing of non-barcoded fragment libraries Non-multiplex paired-end sequencing of RNA libraries Multiplex sequencing of barcoded fragment libraries or RNA libraries (Barcodes 1 to 16) Multiplex sequencing of barcoded fragment libraries or RNA libraries (Barcodes 1 to 96) Sequencing of reverse (F5-P2) tag of non-barcoded fragment libraries Sequencing of reverse (F5-BC) tag of barcoded fragment libraries or RNA libraries 2 SOLiD TM 4 System User Bulletin

3 Updated workflow for 50/35 bp paired-end sequencing Components of the SOLiD 4 Paired End Starter Pack v2 (PN ) Part number Product description Units SOLiD Fragment Library Construction Kit with Size Selection Gels Reagents and gels for 10 fragment libraries SOLiD Fragment Library Oligos Kit Oligos for 10 fragment libraries SOLiD Library TaqMan Quantitation Kit Reagents for 10 libraries SOLiD Pre Deposition Kit 10 reactions SOLiD XD Slide & Deposition Kit v2 8 slides SOLiD ToP Paired End Sequencing Kit DNA Frag. Lib., MM50/35 Reagents for 1 slide SOLiD ToP Instrument Buffer Kit 110 cycles SOLiD ToP Workflow Analysis Reagents Reagents for 2 slides Updated workflow for 50/35 bp paired-end sequencing Paired-end sequencing for DNA libraries Load reagents and slide onto SOLiD Analyzer Run F5-P2 Tag Primers A to E (up to 7 cycles per primer) Paired-end sequencing for RNA libraries Load reagents and slide onto SOLiD Analyzer Run F5-BC Tag Primers A to E (up to 7 cycles per primer) Multiplex paired-end sequencing for DNA or RNA libraries Load reagents and slide onto SOLiD Analyzer Run BC Tag Primers A to E (1 or 2 cycles per primer) Instrument pauses Instrument pauses Instrument pauses Load reagents onto SOLiD Analyzer Run F3 Tag Primers A to E (up to 10 cycles per primer) Load reagents onto SOLiD Analyzer Run F3 Tag Primers A to E (up to 10 cycles per primer) Load reagents onto SOLiD Analyzer Run F5-BC Tag Primers A to E (up to 7 cycles per primer) Instrument completes the run Instrument completes the run Instrument pauses Load reagents onto SOLiD Analyzer Run F3 Tag Primers A to E (up to 10 cycles per primer) Instrument completes the run SOLiD TM 4 System User Bulletin 3

4 Protocol changes Protocol changes Refer to the Applied Biosystems SOLiD 4 System Instrument Operation Guide (PN , Rev. B, 04/2010). Install reagent strip(s) on the instrument Create a sequencing (non-multiplex) run record Page 45, step 2: Refer to the Applied Biosystems SOLiD 4 System Product Selection Guide (PN ) or the Applied Biosystems SOLiD 4 System Instrument Operation Quick Reference Card, Update: SOLiD ToP Paired End 50/35 bp Sequencing Kits (PN ) to determine which reagent strips should be used for a sequencing run. Page 47, step 5: Refer to the Applied Biosystems SOLiD 4 System Instrument Operation Quick Reference Card, Update: SOLiD ToP Paired End 50/35 bp Sequencing Kits (PN ) for the appropriate location of reagent strips on the chiller block. Page 72, step 2f: A typical read length for the F5-P2 tag or F5-BC tag is 35 bases. Create a multiplex sequencing run record Page 77, step 3h: A typical read length for the F5-BC tag is 35 bases. 4 SOLiD TM 4 System User Bulletin

5 Protocol changes Start the sequencing run Page 86, step 1: Minimum disk space for images and results of a paired-end sequencing run Run type Minimum space needed for images Minimum space needed for Primary Analysis results (spch, csfasta, QV.qual) Minimum space needed for Secondary Analysis results (mapping/ pairing) Paired-end (50/35 bp) Multiplex pairedend (50/35/5 bp) Multiplex pairedend (50/35/10 bp) 6.5 TB 3.1 TB <200 GB 6.9 TB 3.3 TB <200 GB 7.3 TB 3.5 TB <200 GB Recommended fill volumes for on-instrument reagents Page 149, Table 36: Fill volumes for paired-end sequencing with a 35-bp F5-P2 or F5-BC tag F5-P2 Tag F5-BC Tag 1 Flowcell 2 Flowcells 1 Flowcell 2 Flowcells Instrument Buffer 2477 ml 4954 ml 2477 ml 4954 ml Storage Buffer 448 ml 896 ml 448 ml 896 ml Cleave 1 Solution 78 ml 156 ml 78 ml 156 ml Cleave 2.1 Solution 78 ml 156 ml 78 ml 156 ml Reset Buffer 72 ml 145 ml 72 ml 145 ml Ligase Buffer 37 ml 75 ml 37 ml 75 ml Universal Buffer 36 ml 73 ml 36 ml 73 ml Imaging Buffer 68 ml 135 ml 68 ml 135 ml SOLiD TM 4 System User Bulletin 5

6 Protocol changes Times for processes Page 156: Times for paired-end sequencing Process Estimated time Steps Estimated time per primer Page 158: Sequencing Primers 1 & 2 and Ligation (F5-P2/F5-BC) Sequencing Primers 3, 4, & 5 and Ligation (F5-P2/F5-BC) Ligation cycle without priming (F5-P2/F5-BC) ~4 to 5 h Reset Prime Ligate Dark Ligate Capper Enzyme Image Cleave RevPhosphatase ~5 to 6 h Reset Prime Bridge Probe Ligate Dark Ligate Capper Enzyme Image Cleave RevPhosphatase ~3.5 to 4.5 h Ligate Dark Ligate Capper Enzyme Image Cleave RevPhosphatase ~24 to 30 h (7 cycles or 35 bp) ~25 to 30 h (7 cycles or 35 bp) Times for entire processes Process Total runtime Paired-end (50/35 bp) Multiplex paired-end (50/35/5 bp) Multiplex paired-end (50/35/10 bp) ~12 to 14 days ~13 to 15 days ~14 to 16 days The estimated time is for a dual-slide run. Estimated times may vary due to differences in imaging time. 6 SOLiD TM 4 System User Bulletin

7 Discontinued kits Discontinued kits Discontinued SOLiD ToP Paired End 50/25 bp Sequencing Kits Part number Product description SOLiD ToP Paired End Sequencing Kit Frag. Lib., MM35/ SOLiD ToP Paired End Sequencing Kit Frag. Lib., MM50/ SOLiD ToP Paired End Sequencing Kit BC Frag. Lib., MM35/25/ SOLiD ToP Paired End Sequencing Kit BC Frag. Lib., MM35/25/ SOLiD ToP Paired End Sequencing Kit BC Frag. Lib., MM50/25/ SOLiD ToP Paired End Sequencing Kit BC Frag. Lib., MM50/25/ SOLiD ToP Sequencing Kit Frag. Lib., F5-P2 Tag, MM SOLiD ToP Sequencing Kit BC Frag. Lib., F5-BC Tag, MM SOLiD 4 Paired End Starter Pack SOLiD TM 4 System User Bulletin 7

8 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. TRADEMARKS: 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Part Number Rev. A 08/2010 Headquarters 5791 Van Allen Way Carlsbad, CA USA Phone Technical Resources and Support For the latest technical resources and support information for all locations, please refer to our Web site at