Let’s see if I’m trimming data or information…
Quality Trimming
To improve the speed and quality of assembly it makes sense to reduce the amount of data (reads) input to the assembler. However, the trick is to reduce data without losing any information. The first step is using sickle to trim the 3’-ends of reads beads on quality scores and discard reads that don’t meet a length threshold. You can read more about their algorithm here, on their github page.
Digital Normalization
I discussed this in the previous blog post but just to reiterate, with the number of reads I have an assembly would take forever and recurring sequencing errors can break up good contigs.
Results
Control
| Category | Pooled Lanes | Quality Trimming | Digital Normalization |
| Paired-end | 116784668 | 114103196 | 22821658 |
| Orphan | NA | 1252357 | 1624925 |
| Total | 116784668 | 115355553 | 24446583 |
| % of Pool | NA | 98.78 | 20.93 |
Condition 1
| Category | Pooled Lanes | Quality Trimming | Digital Normalization |
| Paired-end | 178456950 | 174412784 | 43012608 |
| Orphan | NA | 1926609 | 4014358 |
| Total | 178456950 | 176339393 | 47026966 |
| % of Pool | NA | 98.81 | 26.35 |
Condition 2
| Category | Pooled Lanes | Quality Trimming | Digital Normalization |
| Paired-end | 118787094 | 115437644 | 10536046 |
| Orphan | NA | 1584056 | 1142605 |
| Total | 118787094 | 117021700 | 11678651 |
| % of Pool | NA | 98.51 | 9.83 |
Condition 3
| Category | Pooled Lanes | Quality Trimming | Digital Normalization |
| Paired-end | 395429292 | 385020860 | 2145684 |
| Orphan | NA | 4954819 | 1420062 |
| Total | 395429292 | 389975679 | 3565746 |
| % of Pool | NA | 98.62 | 0.90 |
The amount that was taken from the Strep treated metagenome is a little bit alarm, especially when you remember that it is a much more diverse community that say Cefoperazone (So it’s not just a byproduct of low species richness…). This sample has a whole order of magnitude less reads left after normalization compared to any of the others. I’m not sure how well this will assemble now, but I can tell more once the job for mapping reads to contigs finishes. This will tell me how much what I assemble looks like what I sequenced. If that’s no good, I’ll compare it to the assemblies without normalization and see if khmer is being too greedy. The samples were sequenced on 2 lanes simultaneously and then pooled so it’s unlikey that both lanes sequenced poorly, especially when all other quality metrics are high.