## General/unclassified¶

Question: How do I identify the correct sequencing platform and approach?

Answer: it’s complicated! See The Opinionated Guide to Sequencing and Assembly.

Question: How do I assemble, call SNPs, and compare genetic elements?

Answers: (1) See The Opinionated Guide to Sequencing and Assembly; (2) We didn’t cover that, but look at mapping-based approaches vs graph-based approaches to variant calling (Cortex, SGA); (3) use Mummer and Mauve to visualize.

Question: Should you do de novo assembly, mapping only, or guided assembly?

Answer: Nobody likes guided assemblies, and mapping is often not an option (because the reference is incomplete).

Question: Can you salvage a run without enough data?

Answer: Not really. At best, the preliminary data can be used to generate or refine a strategy for generating more data (e.g. see @Pop paper).

Question: How can I compare M/F expression levels in my critter?

Question: Should assembly workflows for complex metagenomes with a large fraction of eukaryotes use metagenome-specific tools or eukaryotic tools? Or, perhaps which steps in a workflow for complex metagenomes with a large fraction of eukaryotes should use euk tools and which steps should use metagenomic tools?

Answer: While the assembler choice may be different (see The Opinionated Guide to Sequencing and Assembly) almost everything else (read cleanup, QC, etc) can be pretty similar.

Question: What should cores support?

Answer: Obviously it depends, but your core may (or may not) have expertise in more complex sample prep. For example, mate pair library generation needs experienced cores.

## Technology choice¶

Question: At what read length should we be moving towards OLC assemblers instead of De Bruijn?

There’s no obvious answer; further investigation is needed. But, probably, 500bp - 1kb.

Question: What are the best tools and workflows for eukaryote assemblies? Do they differ according to predicted genome size (e.g. 80Mb nematodes vs. 1GB euk.)?

Question: How different are the workflows aimed at metagenomes (where the desired output is gene contigs) vs whole-genome assembly?

Answer: different assemblers may be useful, and the scaling considerations may be different. See The Opinionated Guide to Sequencing and Assembly. Note that contamination is endemic so it’s probably the case that you have a metagenome anyway :).

Question: I would like to develop/obtain a scalable, cloud-based data anaylsis and visualization pipeline.

Answer: khmer-protocols is one attempt at doing this. Note Galaxy, DNANexus, etc. as well. Visualization is still lacking, although note IGV, Ray Cloud Browser.

## Metagenomes and other mixtures¶

Question: How do we deal with “mixed assemblies” (e.g. an endosymbiont)? Is it better to filter reads or contigs? Does the presence of other sequences compromise assembly?

Answer: Blobology@@ will show you the size of the problem, but is not a solution. If you get good contig assembly (e.g. mapping shows that you’ve got most of the reads), it’s better to filter contaminants out from the contigs because you’ll have more signal; but you may have to filter the reads if you didn’t get a good assembly. Different coverage levels from contamination may mislead single-genome assemblers and screw up your recall. If you have lots of contamination, SPADES, digital normalization, and/or various binning approaches (see CONCOCT) may be useful

Question: How can we assemble genomes from “messy” samples (non-clean tissue, data more like a metagenome)? How can we separate out potential symbionts and/or secondarily abundant microbes?

Question: How does metagenomic assembly collapse (or not) information from homologs between strains/species?

Answer: This is very situation/data specific and cannot easily be answered generally. Note that in general highly similar protein sequences will not have highly similar DNA sequences, and so will not be assembled together; we’re really talking about DNA similarity (16s, repeats).

Question: What can one expect to find based on coverage depth?

Answer: Well, you need coverage. preqc (and maybe khmer) may help diagnose low coverage, highe error rates, etc.

Question: What kinds of contamination might you expect to see, and how do you deal with it?

Answer: See above! More work is needed.

Question: Is there a simple way to remove “kitomics”, it fingerprints?

Question: How can I remove endosymbiont (i.e. Wolbachia) reads from fly and butterfly data?

Answer: see above. no standard protocol.

Question: How can we get alpha-diversity, beta-diversity, key taxa at multiple levels, and functional genes for a variety of pathways?

Answer: phylosift and other such tools can help with all of this. HUMANn has good ways to extract functional genes, if you have the references.

## Genome assembly¶

Question: How closely related are my insects? How can I tell?

Answer: Probably very ;). This is really a genotyping question... you can calculate this with k-mer distributions (talk to Jared), too, but in general this is complicated and not necessarily easy or straightforward.

Question: will inbreeding be useful or necessary?

Answer: Useful, but not necessary (just more  sequencing).

Question: How can we sequence individuals to chromosomes or at least obtain meaningful synteny maps?

Answer: See The Opinionated Guide to Sequencing and Assembly; also you may want to use optical maps, linkage maps, and long-read technology.

Question: How do we assemble Eukaryotes? Do we need to use multiple sequencing technologies? Can we use whole-genome amplification? Should we try to make new strain/haplotype-specific references? How about iteratively improving the reference genome with new sequences? How can we make population-specific “type” references? And what exactly is finishing, anyway? Also, do we need to worry about mapping bias?

Answer: For the first two, see The Opinionated Guide to Sequencing and Assembly. Try to avoid whole-genome amplification. There are no good tools available for making either a new reference genome or a “reference graph” (which is really what you want) but Titus may be working on this in the future. You can use several different tools for merging assemblies, improving references, etc – PBJelly with PacBio, GAA and other assembly merging tools, etc. But they don’t necessarily work that well. As for finishing, nobody really does it. Finally, yes, you do need to worry about allelic mapping bias – see @@Wittkopp paper on RNAseq, etc.

## Assembly outcomes, metrics & evaluation¶

Question: do we care about the difference between a 150 contig and a 100 contig assembly? What about 10 or 1000 contigs?

Answer: Well, it depends on your biological question. Generally, outcomes will be limited by your data and require careful planning; see The 10+ Commandments of Assembly.

Question: can I tell how large my genome is from the data?

Answer: preqc can tell you this, and khmer can estimate this (albeit badly).

Question: What do I need to do to improve genome quality?

Answer: that’s more or less the topic of this entire workshop ;)

Question: Can we still use bad-quality data?

Answer: All data is to some extent bad quality data, so yes :). But it will limit the strength of your conclusions (see: ‘science’). More usefully, data is randomly bad, you can work past it; if it’s unknown systematic error, then more data is worse, and you’re in trouble.

Question: What are good quality/completion metrics?

Answer: See “finishing”, above. Also: reads mapped back; marker genes; cegma; REAPR; FRCbam; visual inspection; bridgemapper.

Question: What quality genome assembly can I expect?

Answer: preqc can help tell you this. Don’t work on wheat, soil, or fish, though.

Question: What’s a good way to evaluate completeness of your assembly?

Answer: (1) Mapping of reads. A high percentage is good, and tells you if there is room for improvement. “Treat your assembly as a hypothesis and see how well your reads match.” (2) Bring in orthogonal evidence (see Our list of good practices in (meta)genome assembly).

## Sample prep¶

Question: How really bad is Nextera (insertion bias and dual mode insert size) compared to TruSeq?

Answer: It can be quite a bit worse, or not. You just need to be aware that Nextera is messy in different ways (we need to strong arm Nick into writing a blog post!) Nextera is cheaper and easier, but has insertion bias and requires PCR. High GC organisms are worse with Nextera, and Illumina overall...

Question: how much is it worth striving for PCR-free library preps to avoid amplification bias? and/or going for single-molecule sequencing?

Answer: Depends on details: GC, your question, amount of DNA, etc. Right now PacBio (and Moleculo?) require a lot of input DNA, for example.

#### Previous topic

Thoughts on sequencing strategy

#### Next topic

Predictions

LICENSE: This documentation and all textual/graphic site content is licensed under the Creative Commons - 0 License (CC0) -- fork @ github. Presentations (PPT/PDF) and PDFs are the property of their respective owners and are under the terms indicated within the presentation.

Development and posting of this material, and the associated workshop, were supported by Grant Number R25HG006243 from the National Human Genome Research Institute and an NSF OCI supplement to NSF DBI-0939454.

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