Genome Sequencer FLX System (454 technology)
Genome Sequencer FLX System - Technology Overview Presentation454 Life Science website contains very useful info on GS FLX - Applications - Application Notes - Publications
Using FGCZ NGSequencing Services
FLX Samples Submission form and detailed sample requirements
Quantitate the samples by fluorometry using the Quant-iT DNA assay kit (Invitrogen), following the manufacturer's instructions.Although various quantitation methods are possible, fluorometry has been found to be highly reproducible and to provide consistent emPCR and sequencing results. It is especially important to accurately determine the concentration of individual Amplicon libraries before being pooled prior to emPCR. For instance, if a user is interested to monitor multiple targets together, the determination of an accurate concentration will ensure an even representation of all the targets in the sequencing reaction.
GS FLX technology supports
- Shotgun available with Multiplex Identifiers (up to 12 MIDs)
- Long Span Paired-End Tags with 3 kb, 8 kb and 20 kb span - see Roche Paired-End Library Recommendations and TCB No. 008-2009
- Amplicon - see Guidelines to Amplicon Sequencing
Sequencing Throughput
The tables below list the approximate sequencing throughput for each loading region, and the corresponding total throughput for PicoTiterPlate (PTP).Titanium kits (400 bp reads)
Loading Region Size | # Regions per PTP | Run Throughput
(Mbp) | ||
per Region | per PTP | |||
Large | 2 | 180-280 (450-650 k reads) | 360-560 | |
Medium | 4 | 60-110 (160-250 k reads) | 240-440 | |
M/S | 8 | 30-55 (80-120 k reads) | 240-440 | |
Small | 16 | 10-20 (25-40 k reads) | 160-320 |
XL+ kits (700 bp reads)
Loading Region Size | # Regions per PTP | Run Throughput
(Mbp) | ||
per Region | per PTP | |||
Large | 2 | 250-400 (450-650 k reads) | 500-800 | |
Medium | 4 | 100-160 (160-250 k reads) | 400-640 | |
M/S | 8 | 40-70 (80-120 k reads) | 320-560 | |
Small | 16 | 16-25 (25-40 k reads) | 240-400 |
GS FLX Titanium Series Transcriptome Sequencing.
cDNA normalisation is important for de novo trancriptome sequencing using the Genome Sequencer FLX System. It essentially increases the efficiency of transcriptome sequencing and functional sequencing. As cDNA normalisation results in equalisation of the abundance of different transcripts and increase in the number of previously non-detectable genes in cDNA samples.The FGCZ recommends the use of two kits from Evrogen:
First, the MINT cDNA synthesis kit Cat SK001.
Followed by the TRIMMER cDNA Normalization kit Cat NK001.
An important note regarding the use of the MINT cDNA synthesis kit for 454 sequencing is that modified primers are required.
The 1ul of 3'primer (10uM) required in the kit is substituted by 1ul of PolTdeg (10uM).
The 2ul of the PCR Primer M1 (10uM) are substituted by 1ul of M1ACGG (10uM) and 1ul of polTM1 (10uM).
See sequences of the cDNA synthesis primers for 454 sequencing.