Our highly specialized software is able to process datasets from all kinds of complex scenarios, detect lab protocols and remove their adapters, artefacts, MIDs along with technology-specific artefacts. Although it was developed originally for Roche 454 platform, some lab procedures are independent of the sequencing technology used later and thus we can fix also datasets from other sequencers (IonTorrentPGM, IonProton , Illumina).
The pricing is calculated individually due to vast differences in computational times between various categories and their combinations. Below is a listing of key properties of various processing approaches and it is aimed to serve as a Checklist.
A. Pre-processing protocols (irrelevant from the following sequencing technology):
We can correct reads from samples pre-processed by any of these assays not only in conjunction with Roche 454 but also with IonTorrent and Illumina.
□ [[NONE]] Tick this item when the sample contains only sequencing-technology-specific adaptor sequences, when there is nothing introduced during sample pre-processing (so no extra work for us!).
For example, this is the case if you just nebulized / sonicated / sheared genomic or mitochondrial DNA, or fragmented sample using dsDNA endonuclease (Fragmentase®) method, NEBNext®, NewEngland BioLabs (Adey et al., 2010).
Contrary to the above “NONE” item, the alternatives listed below introduce additional nucleotide sequences into sequencing reads, and thy are independent of the sequencing technology.
□ [[a]] Roche transcriptome cDNA preparation
□ [[b]] non-normalized cDNA, non-oriented insert sequences
□ [[c]] non-normalized cDNA, non-oriented insert sequences
□ [[d]] non-normalized cDNA, oriented insert sequences
Clontech SMART, SMARTer and SMARTerII protocols:
□ [[e]] non-normalized cDNA, non-oriented inserts
□ [[f]] non-normalized cDNA, oriented inserts
□ [[g]] normalized cDNA, non-oriented inserts
□ [[h]] normalized cDNA, oriented inserts
□ [[i]] modified Clontech PlugOligo with MmeI(Clontech SMARTTM) (Zeng et al., 2011)
□ [[j]] non-normalized cDNA, non-oriented inserts, modified MINT_PCRprimer_M1 with MmeI (Brenchley et al., 2012)
More tweaks of the Clotech’s method:
□[[k]] Cap-Trsa-CV-based normalized cDNA method (modified Clontech SMART by J. Buchanan-Carter, Z. Smith, K. Mockaitis, M. Matz)
Other sample pre-processing protocols:
□ [[l]] ncRNA sample (Huttenhofer and Vogel 2006; Mrazek et al., 2007)
□ [[m]] microRNA sample
□ [[n]] T7 RNA polymerase promoter-based adapter assays (Clontech GenomeWalker-based cDNA reads (PT3042-1, PT1116-1, a method developed at LGC Genomics, Berlin)
□ [[o]] SP6 polymerase-based in vitro run-off transcripts
□ [[p]] transposase-based adaptor insertion, Nextera, Epicentre® (Adey et al., 2010)
□ [[q]] BAC clone sequencing
□ [[r]] I.M.A.G.E. clone sequencing
□ [[s]] a GATC Biotech, Konstanz protocol (“pine tree” cDNA project method), (Chang et al., 1993; Pavy et al., 2008)
□ [[t]] Suppression subtractive hybridization (SSH) PCR (Clontech PCR-Select Bacterial genome subtraction kit) (Diatchenko et al., 1996)
□ [[u]] full-length cDNA method (BioS&T uncloned normalized cDNA, possibly following the original F. Bonaldo method)
□ [[v]] Nimblegen capture method (Bashiardes, 2005)
□ [[w]] custom oligo-dT adapter with GsuI restriction site (Leroux et al., 2010; Le Cam et al., 2012)
In the above listing several protocols are deemed to exist based on our experience with the publicly available data. Unfortunately, we do not know yet their proper names nor publication status and not even their author names. In any case, we can do more then we advertise here. Just email us your experimental setup.
B. Sequencing Protocols and their alternatives, incl. IonTorrentPGM:
Roche (454TM) Protocols and their alternatives
Standard Amplicon (or Paired-end) Protocols:
GS20/GS FLXTM emPCR Kit II (aka amplicon A, Paired End) (Roche Application Notes No. 3 (2006) and No. 8 (2007), Kappa Biosystems #KP0001):
□ [[1]] amplicon A without GSMIDs
□ [[2]] amplicon A with GSMIDs on the left
□ [[3]] amplicon A with GSMIDs on both sides*
GS20/GS FLXTM emPCR Kit III (aka amplicon B) (Jarvie and Harkins (2008), Galan et al. (2010), Kappa Biosystems #KP0001):
□ [[4]] amplicon B no GSMIDs
□ [[5]] amplicon B with GSMIDs on the left
□ [[6]] amplicon B with GSMIDs on both sides*
GS20/GS FLXTM emPCR Kit II+III combined (amplicon A+B):
□ [[7]] amplicon A+B without GSMIDs
□ [[8]] amplicon A+B with GSMIDs on the left
□ [[9]] amplicon A+B with GSMIDs on both sides*
Titanium Amplicon (or Paired-end) Protocols:
GS FLX TitaniumTM emPCR Kit II (aka amplicon A, Paired End):
□ [[10]] amplicon A without TiMIDs
□ [[11]] amplicon A with TiMIDs on the left
□ [[12]] amplicon A with TiMIDs on both sides
GS FLX TitaniumTM emPCR Kit III (aka amplicon B):
□ [[13]] amplicon B without TiMIDs
□ [[14]] amplicon B with TiMIDs on the left
□ [[15]] amplicon B with TiMIDs on both sides*
GS FLX TitaniumTM emPCR Kit II+III combined (amplicon A+B):
□ [[16]] amplicon A+B without TiMIDs
□ [[17]] amplicon A+B with TiMIDs on the left
□ [[18]] amplicon A+B with TiMIDs on both sides*
Whole Genome Shotgun Protocols:
GS20/GS FLX Standard (prepared using emPCR Kit I)
□ [[19]] GS20/GSFLXstd without GSMIDs
□ [[20]] GS20/GSFLXstd with GSMIDs on both sides*
Rapid Library Y-type adapter for Taqman MGB qPCR quantification (Zheng et al., 2010)
□ [[21]] Y-type-GS20-FLXstd-Taqman-MGB_qPCR without GSMIDs
□ [[22]] Y-type-GS20-FLXstd-Taqman-MGB_qPCR with GSMIDs on both sides*
Rapid Library Y-type adapter for Taqman MGB qPCR quantification (Zheng et al., 2011)
□ [[23]] Y-type-RapidLib-Taqman-MGB_qPCR without GSMIDs
□ [[24]] Y-type-RapidLib-Taqman-MGB_qPCR with GSMIDs on both sides*
GS FLX/FLX+ Titanium (emPCR Kit I) (Roche TCB-004 2009)
General Library Preparation Protocol with Roche adapter
□ [[25]] no TiMIDs (IonTorrentPGM shotgun data also belong here)
□ [[26]] with TiMIDs on the left
□ [[27]] with TiMIDs on both sides*
Rapid Library Preparation Protocol with Roche adapter
□ [[28]] Y-type-RapidLib without RLMIDs
□ [[29]] Y-type-RapidLib with RLMIDs on both sides
Rapid Library Preparation Protocol with oligonucleotide adapter(s) from http://IDT.dna.com:
□ [[30]] Y-type-RapidLib-IDT without RLMIDs
□ [[31]] Y-type-RapidLib-IDT with RLMIDs on both sides
Crazy datasets:
□ [[32]] mis-carried samples with two Roche adapters surrounding the sample sequence
□ [[33]] mis-carried samples with two Roche adapters surrounding the sample sequence with GSMID/TiMID/RLMID tags
□ [[34]] mixed reads with two or more different Roche adapters in same sequencing region
□ [[35]] mixed reads with two or more different Roche adapters in same sequencing region with GSMID/TiMID/RLMID tags
IonTorrent / IonProton Protocols
At least some Ion protocols use same adapters like the original 454 (Roche) General Library Preparation Protocol (emPCR kit I). So far we saw these datasets to come from IonTorrentPGM machines with either 260 or 520 flows. Please refer to item [[25]] in the above listing.
Illumina Protocols
Transcriptomic datasets based on Clontech SMART or Evrogen MINT protocols are supported, possibly other like fl-cDNA, etc. We can remove Illumina adapters as well in an additional step.
Notes:
* Analysis of multiplexed samples is computationally highly expensive. It is getting even worse once you have different MIDs on the left and right end of the read. However, the true performance killers are Evrogen-based transcriptomic setups involving MIDs and datasets with doubled Roche or 3rd-party adapters. For a more thorough pricing calculation please contact us with details of your experimental setup.
For example, tick:
[[NONE]] + [[25]] for shotgun data from a General Library Prep.,
[[a]] + [[28]] for Roche transcriptomic dataset processed through the Rapid Library Protocol,
[[f]] + [[25]] would be an example of a normalized Evrogen MINT2 (Clontech SMART) approach followed by General Library Prep.,
[[NONE]] + [[10]] for paired-end reads from a General Library Prep.