lib-reamp_eecseq.Rmd

---
title: "Library Reamplification"
params:
  num_seq_lib: 1
---
```{r setup, include=FALSE}
library(tidyverse)
library(knitr)
```

### Library re-amplification
KAPA HiFi HotStart ReadyMix is a pre-made master mix that contains the polymerase, buffer, dNTPs, etc already.
```{r}
reamp <- tribble(~item, ~quantity,
                 "kapa_hifi_hotstart_readymix_ul", 12.5 * params$num_seq_lib,
                 "f_p1_primer_universal_working_ul", 1.25 * params$num_seq_lib,
                 "r_p2_primer_illumina_index_working_ul", 1.25 * params$num_seq_lib, 
                 "tips_100", 1,
                 "tips_10", 3 * params$num_seq_lib,
                 # make room for small or large numbers of samples
                  ifelse(params$num_seq_lib <20, "tubes_200ul", "plates"), ifelse(params$num_seq_lib < 20, params$num_seq_lib * 2, ceiling(params$num_seq_lib/96) * 2))
```
- Assemble each library amplifcation reaction as follows
```{r}
kable(tribble(
  ~component, ~volume,
  "KAPA HiFi HotStart ReadyMix (2X)", "12.5 ul",
  "PCR1_P5 Primer (Universal)","1.25 ul",
  "PCR2 Primer (Indexed)", "1.25 ul",
  "Enriched Library", "10.0 ul",
  "Total Volume","25 ul"))

```

**NOTE:** It's important to use the same INDEX primer as the original library prep!

- Mix thoroughly and centrifuge briefly.
- Amplify using the following cycling protocol:
```{r}
kable(tribble(~Step,~Temp,~Duration,~Cycles,
"Initial denaturation", "98 °C", "45 sec", "1",
"Denaturation","98 °C", "15 sec", "12",
"Annealing", "60 °C", "30 sec", "12",
"Extension", "72 °C", "30 sec", "12",
"Final Extension", "72 °C", "1 min", "1",
"Hold", "4 °C ", "∞", "1"))
```