---
# System prepended metadata

title: mEleMax1
tags: [VGP, Elephant Project]

---

---
title: 'mEleMax1'
---

*Asian elephant* VGP Genome
===

###### tags: `Elephant Project` `VGP`

:::construction::: UNDER CONSTRUCTION :::construction:::


Table of Contents
---
[TOC]


Aim
---
:::info
**Generate a VGP genome for the Asian elephant using the last available version of the assembly pipeline (2.0+)**
:::


Data
---

* ***Transcriptomic data for annotation***

Total RNA-Seq (TruSeq total RNA protocol) with rRNA depletion using baits. HiSeq4000?? PE 101 bp 

|Sample ID|Paired reads #|Total throughput (Gbp)|
|:-:|:-:|:-:|
|1_291_Lung|203758250|41.1|
|2_445_thyroid|196528506|39.7|
|3_445_lymphnode|174584352|35.2|
|4_445_salivarygland|162864238|32.9|
|5_466_ovary|181593456|36.7|

:warning: Samples *2_445_thyroid*, *3_445_lymphnode* and *4_445_salivarygland* show not ideal GC distribution (among other things):
![](https://i.imgur.com/KBj2ap8.png)
![](https://i.imgur.com/yIx9IHA.png)
![](https://i.imgur.com/pQCcZjp.png)
  
:mag_right: Ribosomal rRNA was extracted by mapping against 131 sequences from [RNAcentral](https://rnacentral.org/) database obtained using the query filter:
```
Loxodonta africana AND so_rna_type_name:"RRNA" AND rna_type:"rRNA" AND qc_warning_found:"False" AND so_rna_type_name:"Cytosolic_rRNA" AND TAXONOMY:"9785"
```
:+1: Low amount of rRNA found:

```csvpreview {header="true"}
Sample ID,Paired reads #, %
1_291_Lung,"967,478", 0.474
2_445_thyroid,"426,906", 0.217
3_445_lymphnode,"746,815",0.427
4_445_salivarygland,"511,723",0.314
5_466_ovary,"495,435",0.272
```

* ***Genomic data***

Male asian elephant (origin: India)
2n=56 (27+1)

To get available data:

```
mkdir -p ~/mEleMax1/genomic_data/pacbio
aws s3 cp s3://genomeark/species/Elephas_maximus/mEleMax1/genomic_data/pacbio_hifi/ ~/mEleMax1/genomic_data/pacbio_hifi --recursive --no-sign-request
mkdir -p ~/mEleMax1/genomic_data/bionano
aws s3 cp s3://genomeark/species/Elephas_maximus/mEleMax1/genomic_data/bionano/ ~/mEleMax1/genomic_data/bionano --recursive --no-sign-request
mkdir -p ~/mEleMax1/genomic_data/
ws s3 cp s3://genomeark/species/Elephas_maximus/mEleMax1/genomic_data/arima/ ~/mEleMax1/genomic_data/arima --recursive --no-sign-request
```

:::info
HiFi total Gbp: 171.14
HiFi **raw** coverage: ~53x
:::

|Sample Name|% Dups|% GC|Length|M Seqs|
|:-|:-:|:-:|:-:|:-:|
|m54306U_210321_092024.Q20|0.4%|40%|16,355 bp|0.5|
|m54306U_210421_201740.Q20|0.4%|40%|12,485 bp|0.4|
|m54306Ue_210627_165734.hifi_reads|0.3%|40%|10,374 bp|0.3|
|m54306Ue_210705_134503.hifi_reads|0.4%|40%|8,033 bp|0.4|
|m64055_210419_095605.Q20|0.6%|41%|16,152 bp|0.8|
|m64055_210425_003744.Q20|0.5%|40%|10,970 bp|0.7|
|m64330e_211019_195149.hifi_reads|1.4%|41%|15,931 bp|1.9|
|m64334e_211022_161721.hifi_reads|1.4%|41%|16,119 bp|2.1|
|m64334e_211024_014441.hifi_reads|1.4%|41%|16,346 bp|2.1|
|m64334e_211025_111049.hifi_reads|1.3%|41%|15,972 bp|2.0|


Results
---

:mag: All scripts used during the assembly are available on [**GitHub**](https://github.com/diegomics/VGP_Asian_elephant)


:black_circle: **_QC_**

:::success
**PacBio HiFi** quality looks GOOD! :slightly_smiling_face:
- Only 0.1% of reads were discarded during trimming
:::

---

![](https://i.imgur.com/SR53hdI.png)



```csvpreview {header="false"}
Estimated genome size (bp),"3,350,136,551"
Transition parameter*,36
Maximum read depth,108
```
*transition between haploid and diploid coverage depths

:::success
Genome size estimation is **very** reasonable
:::


:black_circle: **_Contig assembly (hifiasm) & Purge haplotigs and overlaps (purge_dups)_**

:::info
:bulb: Based on extensive testing with this data, maybe a good and flexible way to face the contigging stage is to test the different **purging duplicates options** within hifiasm (none `-l0`, light `-l1`, aggresive `-l2`, and aggresive - high heterozigosity rate `-l3`) and posteriorly run purge_dups, all with VGP suggested cutoffs (i.e., *Maximum read depth* for the **coverage upper bound** `--purge-max` in hifiasm, and *Transition parameter* and *Maximum read depth* for `-m` and `-u` in purge_dups, respectively)
:::

:point_right: Best results were obtained with `l2` and `l3` options (see full results during options testing [here](https://hackmd.io/_mfIxNV4QYGnF5HD9LAUJA))

![](https://i.imgur.com/8jfTt4R.png)


* purge_dups cutoffs histograms for p1 and q2

**mEleMax1_l2**
![](https://i.imgur.com/J2BbO4X.png)

**mEleMax1_l3**
![](https://i.imgur.com/yca8iEC.png)



* K-mer Multiplicity of c1c2 and p1q2 (stacked)

**mEleMax1_l2**
![](https://i.imgur.com/MJmgfyl.png)


**mEleMax1_l3**
![](https://i.imgur.com/yvuwd0s.png)


* QV Score (detailed)
![](https://i.imgur.com/ljJAvU9.png)

* Kmer Completeness (detailed)
![](https://i.imgur.com/bVCxhPi.png)



* BUSCOv5 database: mammalia (detailed)


**mEleMax1_l2 p1**
```
C:96.1%[S:95.3%,D:0.8%],F:1.1%,M:2.8%,n:9226
8861    Complete BUSCOs (C)
8789    Complete and single-copy BUSCOs (S)
72      Complete and duplicated BUSCOs (D)
105     Fragmented BUSCOs (F)
260     Missing BUSCOs (M)
9226    Total BUSCO groups searched
```
**mEleMax1_l3 p1**
```
C:96.1%[S:95.3%,D:0.8%],F:1.1%,M:2.8%,n:9226
8862    Complete BUSCOs (C)
8789    Complete and single-copy BUSCOs (S)
73      Complete and duplicated BUSCOs (D)
105     Fragmented BUSCOs (F)
259     Missing BUSCOs (M)
9226    Total BUSCO groups searched
```



:::success
Assemblies with either `-l2` and `-l3` hifiasm options look **amazing**
Less than 120 contigs: considering that n=28, we may have like 4 contigs per chromosome :exploding_head:
Let's continue with both in parallel :muscle:
:::

:black_circle: **_Bionano scaffolding_**

:::info
There was no removal of Trim trailing N-bases in the scaffolded assembly (apparently there are none)
:::

![](https://i.imgur.com/0BC78yq.png)


* BUSCOv5 database: mammalia (detailed)


**mEleMax1_l2 s1**
```
C:96.0%[S:95.2%,D:0.8%],F:1.1%,M:2.9%,n:9226
8860	Complete BUSCOs (C)
8785	Complete and single-copy BUSCOs (S)
75	Complete and duplicated BUSCOs (D)
101	Fragmented BUSCOs (F)
265	Missing BUSCOs (M)
9226	Total BUSCO groups searched
```
**mEleMax1_l3 s1**
```
C:96.0%[S:95.2%,D:0.8%],F:1.1%,M:2.9%,n:9226
8859	Complete BUSCOs (C)
8785	Complete and single-copy BUSCOs (S)
74	Complete and duplicated BUSCOs (D)
103	Fragmented BUSCOs (F)
264	Missing BUSCOs (M)
9226	Total BUSCO groups searched
```

:::success
Assemblies looking very good so far. Very good contiguity and BUSCO parameters within what is seek. Hopefully, HiC data will allow a good final result.
:::

:black_circle: **_HiC scaffolding_**

:::info
:bulb: Based on testing, l3 option ended being the best 
:::

![](https://i.imgur.com/PBGYh5c.png)

![](https://i.imgur.com/CNx3WrR.png)
