008-20 GS FLX Technical Bulletin No. 09 TCB No. 008-2009 May 2009 Technical Bulletin Genome Sequencer FLX System Multi Span Paired End Libraries: Guidelines and Additional Information Summary This bulletin provides users of Multi Span Paired End libraries with information and guidance and is intended for use as a supplement to the Method Manuals and Quick Guides. The content is divided into two sections: Usage guidelines and coverage estimates for the various paired end library types Tips and tricks regarding the laboratory workflow 2009 454 Life Sciences Corp. All rights reserved. 454, 454 SEQUENCING, Genome Sequencer, GS FLX Titanium, PicoTiterPlate, PTP, and empcr are trademarks owned or used by 454 Life Sciences Corp., a Roche Company Branford, CT, 06405 USA Page 1 of 8 Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim, Germany Roche Diagnostics Corp. Roche Applied Science Indianapolis, IN, 46250 USA
Note: This bulletin is intended to serve as an addendum to the existing GS FLX Titanium 3 kb and GS FLX Titanium 20 kb and 8 kb Span Paired End Library Preparation Method Manuals. This document alone does not provide sufficient instructions to use the GS FLX Titanium Paired End Adaptor Set. Relevant sections of the Method Manuals are referenced in this document. As similar procedures exist in both Method Manuals, only one document, GS FLX Titanium 20 kb and 8 kb Span Paired End Library Preparation Method Manual is referenced in the interest of conciseness. Paired End Library Usage Guidelines Introduction Multi Span Paired End libraries provide the user with a powerful tool for a variety of applications including de novo genome assembly. However, an understanding of the characteristics of the various library types is critical to ensure efficient library generation and subsequent sequencing. One important consideration is the number of unique molecules in a given library. While this cannot readily be determined precisely for a given library, general guidelines can be provided. This information will allow for estimation of the number of Runs of a given library type that might be required to assemble a genome of a certain size. Importantly, these are intended to be guidelines only, as individual genome composition and complexity, not merely genome size, impact the type and depth of coverage required for de novo genome assembly. Generating Multiple Libraries It is possible to generate multiple, independent libraries from the amount of genomic DNA specified as starting material for each protocol as outlined in Figure 1. Generally, sufficient material exists after the Fill-In Reaction (Step 3.5) for at least two independent DNA Circularization reactions (Step 3.6). In addition, only half of the Adapted Paired End library bead suspension volume is employed in the Library Amplification (Step 3.10); so, two amplifications are possible. Typically four independent amplified libraries can be made from a single input genomic DNA sample and in the case of the 3 kb library protocol, often twice as many (eight). The ability to make multiple independent libraries from the recommended amount of starting material plays an important role in considering your library and Run budget for de novo genome sequencing, particularly for larger genomes. Figure 1. Diagram of Protocol Split Points for Multi Span Paired End Libraries Page 2 of 8
Genome Assembly Coverage Recommendations For each library span distance, Table 1 outlines the parameters that should be used as a starting point when considering de novo genome sequencing experiments with the 454 Sequencing System. Often multiple spans plus shotgun reads are required and the optimal blend of these depends on genome composition and complexity. For many, particularly smaller (> 5 Mb) microbial genomes, a single 8 kb Span Paired End Library may be sufficient to yield a high quality assembly. Larger or more complex microbial genomes may benefit from a mix of shotgun and 3 or 8 kb libraries for a total of 15X coverage. For more complex genomes a blend of 12X shotgun, 3X 3 kb pairs, and 2X of 20 kb (and potentially 2X of 8 kb) pairs is a good starting point. In each of these cases, X refers to absolute sequencing yield (bases) compared to the genome size. Using the above example, 3X coverage of a 250 Mb genome with 3 kb pairs would be 750 Mb or 1.5 to 2 Titanium series Runs. Further information on this important topic will be made available shortly via webinars or additional documentation. Table 1. Coverage Guidelines for Multi Span Paired End Libraries It is critical to consider the number of unique fragments in a given library. It is this value, relative to the physical coverage desired for assembly of a genome of a given size, that helps determine how many libraries should be made and sequenced. For example, experience suggests that a given 3 kb Span Paired End library may be efficiently sequenced with up to two full (2-region) GS FLX Titanium series Runs as shown in the table above. This will generally yield sufficient coverage of fragments with this span distance for the assembly of a 250 Mb genome. In the case where eight independent 3 kb libraries may be made from 5 μg starting material, it logically follows that a 2 Gb genome (250 Mb 8) could be covered at the recommended depth for assembly with the GS De Novo Asssembler package. To avoid a high level of redundant sequencing due to library oversampling, it is essential to employ these recommendations when planning sequencing experiments. In particular, the Runs per Library should be considered as a maximum value. One approach to prevent oversampling is to monitor redundancy following sequencing. This may readily be assessed by considering the first 100 bp of each read and monitoring how often the same motifs are repeated. Note: It is not generally efficient to sequence 20 kb libraries in more than one region of a 2- region GS FLX Titanium sequencing Run. Sequencing additional, independent libraries (per Table 1 up to four may be made per 30 μg of input DNA) is recommended for genomes larger than 100 Mb. If your final 20 kb library quantitation suggests a yield of less than 2x10 10 molecules, it is recommended to sequence the library using no more than one region of a 4- region GS FLX Titanium sequencing Run to avoid oversampling of the library. Page 3 of 8
Workflow Tips and Precautions General When preparing multiple libraries in parallel, master mixes of reagents may be made for all reactions. As with other processes, preparing additional volume (i.e. 10% more than what is required) is advisable to avoid having insufficient volume for distribution to all samples. Mix all reactions gently, but well, to ensure proper and complete mixing of all reagents and reactions. All incubation steps with reaction temperatures at or below 50 C may be performed in a heated block. Any incubations above 50 C should be performed in a thermocycler with a heated lid to prevent evaporation/condensation of reaction components during the incubation. In the SPRI bead calibration procedure (Section 5.5), prior to performing the DNA 7500 chip assay, it is important to set the BioAnalyzer 2100 Height Threshold to value= 5, in the assay properties tab in the Expert 2100 software to obtain accurate resolution of the peaks. As described in the Method Manual, the 10x PNK buffer must be fully in solution prior to use. If any precipitate is observed after thawing, resuspend via brief (1-3 min.) heating in a 37 C heat block combined with vortexing. The process should be repeated until all material is in solution. Input DNA (Section 2.2.1) Starting with high quality input material is critical for success with these methods. Employing genomic DNA of the prescribed attributes will consistently yield better results than degraded or impure material. Please note that DNA quantity must be assayed using fluorometric methods. Quantification via UV absorption is often inconsistent and can be substantially influenced by a variety of factors. While these methods are sufficient for other protocols, their use is not recommended for the preparation of Paired End or other library types in the Genome Sequencer FLX System. 3.1 DNA Fragmentation During the DNA Fragmentation step you may observe bubbles in your sample while it is in the HydroShear syringe. These bubbles may be removed by turning the valve to a position between the Input and Output settings while the syringe volume is being compressed. This procedure is described in further detail in the HydroShear user manual. Caution: Remember to turn the valve to the Output position before the HydroShear aspirates If there are bubbles in the HydroShear after aspirating your sample, flick the syringe with your finger to get the bubble off of the plunger. Page 4 of 8
3.2 Fragment End Polishing Resuspension of the GenFind Beads in 80 μl of Buffer EB (10 mm Tris-HCl, ph 8.5) should be achieved by manually shaking the solution back and forth over the bead pellet until the beads are completely resuspended. Placing the solution on a rotator is not recommended as this approach can result in incompletely resuspended sample remaining on the beads. 3.3 Circularization Adaptor Ligation As stated in the manual, be sure to mix the contents of the ligation reaction both prior to and after adding DNA ligase. If making a master mix with the ligase components, ligase should not be included and should only be added to the reaction mix after the reaction mix has been added to each sample. 3.4 Library Span Size Selection [8 kb protocol only] Failure to observe a bright band below the 100 bp marker prior to gel extraction in the 8 kb protocol is an indication that the Circularization Adaptor was not added in the previous step (Section 3.3). Do not proceed with the library if you do not observe this band. Note: This bright band below the 100 bp marker will not be seen in the 20 kb Span Paired End Library protocol. When making the gel excision, it is recommended to cut the region of the gel where the DNA is most concentrated (brightest) rather than cutting precisely around the desired fragment size. For example, if a 20 kb Span library is intended, but the majority of the fragments observed during gel extraction are approximately 16 kb, it would be preferable to collect ample material of this size rather than less material centered around the 20 kb target size. This, of course, will create a 16 kb Span Paired End library. When the region excised is smaller or larger than intended, it is also advised to make a back-up cut, below and/or above the first region excised. This gel fragment may be retained at 4 C for later use or processed along with the other region(s) excised if desired. Prior to loading your sample, but after adding the 600 μl of 1X TAE into the Elutrap, examine the Elutrap from the side to ensure that none of the 600 μl volume is leaking out of the trap region. If the solution in the trap region is leaking, tighten the pressure screw further and check again. When adding fresh 1X TAE into the Elutrap to cover the gel slices, be sure not to overload the TAE. The volume added should be just enough to cover the gel slices. Too much 1X TAE will spill over the BT2 membrane and dilute your sample. The BT2 membrane is very fragile; be careful when pipetting your sample out of the trap region. Good results have been obtained using a P-200 pipettor (a P-1000 is too large and will cause the membrane to tear). If you accidentally tear the BT2 membrane, remove no more than 1000 μl from the trap region. The majority of your DNA will be captured in this volume. Be advised that there is an increased chance of sample loss due to dilution if membrane tearing occurs. Page 5 of 8
The time required to concentrate your sample is a factor of volume and concentration when using the Microcon YM-50 filter unit. More concentrated or higher volume samples will take longer to concentrate with the Microcon device. It is preferable to initially underestimate the amount of time to centrifuge your sample to avoid drying out the membrane. Refer to the Microcon manual for estimates on spin times. Possible Sample Loss: It is critical to avoid drying out the Microcon column membrane as this will lead to sample loss. The recommended volume to recover from the Microcon filter unit is between 38 μl and 42 μl. If your recovery volume is less than 38 μl, bring the volume up to 38 μl with Buffer EB (10 mm Tris-HCl, ph 8.5). If your recovery volume from the Microcon is greater than 42 μl, place the sample into the same Microcon, filter side up, and centrifuge to concentrate further. Using a new Microcon column at this point would likely result in a significant loss of your sample. This is also stated in the Microcon user manual. 3.5 Fill-In Reaction Fluorometry-based methods are required for quantifying pre-circularized DNA. As described in the Method Manual, the use of the Quant-it Picogreen (Invitrogen) assay is recommended. We advise using a serial dilution of the supplied standard material from 250 pg to 0.9 pg. A 1:100 or 1:1000 dilution of your pre-circularized DNA sample is typically required to obtain a reading within the range of the standard curve. 3.6 DNA Circularization Add freshly diluted DTT prior to proceeding with exonuclease treatment step. Mix gently and completely prior to adding exonuclease reagents. Stock 1 M DTT can be kept for up to 1 year at -20 C. Once made, the stock should be aliquotted into small volumes to avoid repeated freeze-thaw cycles. After adding all exonuclease reagents, it is critical to mix the solution gently but completely. Gently mixing the samples by inverting and then shaking the solutions by hand for 30 seconds is typically sufficient to completely mix the reaction components. Library Quality: Proper mixing during exonuclease treatment is essential to ensure complete digestion of undesired, linear (uncircularized) DNA. Underperformance at this step may impact the final library yield and/or quality of paired reads obtained from sequencing. Ensure that the supplied Carrier DNA is added after exonuclease treatment as specified in the Method Manual. Substituting any other material for the Carrier DNA is strongly discouraged and will likely result in a reduction of true paired reads obtained from sequencing. 3.7 Circularized DNA Fragmentation (Nebulization) Process the entire volume of your nebulized solution + PBI through a single MinElute column - do not split your sample across multiple columns as this will cause an excessive loss of sample. Page 6 of 8
If the BioAnalyzer DNA 7500 LabChip assay results suggest a larger than desired fragment population (for example, a long right-tailed distribution) repeating nebulization is suggested. Simply bring the sample volume to 100 μl and repeat Step 3.7 after adding the sample to 500 μl of Nebulization buffer in a new nebulizer cup. Some sample loss will be expected but library quality and the yield of true paired reads will be substantially better from a properly fragmented sample. 3.8 Fragment End Polishing (no additional comments) 3.9 Library Immobilization, Library Adaptor Ligation and Fill-In Reaction Once your sample is bound to the immobilization beads, vigorously vortex your samples for 5-10 seconds before each enzymatic reaction (for a total of three times), exchanging the solution with fresh 1X TE each time. This will help remove all residual non-biotinylated DNA. After removing the last TE wash, do an additional quick (5 seconds) dry spin and place the tube back on the MPC. Use a P-10 or similar small volume pipettor to remove the last residual volume of TE prior to proceeding to the next step. 3.10 Library Amplification (no additional comments) 3.11 Final Library Size Selection Make sure you have calibrated your lot of SPRI beads per Section 5.5 of the Method Manual. When measuring the SPRI solutions, both prior to and after mixing with the sample (e.g. supernatant after first SPRI cutoff), make sure all residual solution is expelled from the tip prior to disposing of the tip. See section 5.5, and page 52 in particular of the Method Manual for proper pipetting of SPRI bead solutions. Accurately measuring the solution after the first SPRI cut-off is the most sensitive stage. The solution will always be less than the sum of the sample and the SPRI solution due to the volume of the beads and must be accounted for with EB solution. Library Quality: Accuracy during SPRI size selections is critical for the generation a population of appropriately sized library fragments. Differences of only a few microliters can result in poorly sequencing libraries. During the size selection (section 3.11, step 7), try to measure as few times as possible; overestimating by 2-4 microliters the amount of EB to add to account for the mass of the beads (see previous hint) will help. After removing the last 70% ethanol wash from the beads, perform an additional quick (5 seconds) dry spin and place the tube back on the MPC. Use a P-10 or similar small volume pipettor to remove the last residual volume of ethanol prior to proceeding to the next step. Page 7 of 8
3.12 Paired End Library Isolation (single-stranded) To ensure proper elution of the Paired End DNA library from the MinElute filter, add 10 mm Tris-HCl, ph 7.5-8.5 at ambient temperature and let the buffer sit in the column for 1 minute prior to centrifugation. Fluorometry-based methods are required for quantifying the final single-stranded DNA. As described in the Method Manual, the use of the Quant-it Ribogreen (Invitrogen) assay is recommended. We advise using a serial dilution of the supplied standard material from 250 pg to 0.9 pg. A 1:10 dilution of final library is typically required to obtain a reading within the range of the standard curve. If no library peak is detected on the RNA chip, the library may still be quantifiable. In this case, assume a peak of 600 bp and calculate molecules using this value. Previous results suggest that as long as the total number of calculated library molecules is greater than 2x10 9, the library can be sequenced. However, greater redundancy will likely result from such a library. If sufficient pre-circularization DNA remains, it is advised to repeat the protocol from that step. Page 8 of 8