# Kraken 2/Bracken
### Kraken 2 is the newest version of Kraken, a taxonomic classification system using exact k-mer matches to achieve high accuracy and fast classification speeds. This classifier matches each k-mer within a query sequence to the lowest common ancestor (LCA) of all genomes containing the given k-mer. The k-mer assignments inform the classification algorithm.

## Install Kraken 2 and Bracken:
`conda create -y -n kraken2 -c conda-forge -c bioconda -c defaults kraken2=2.0.9beta bracken=2.6.0`

## Download the database:
`scp -r astrobio@149.165.170.83:/vol_b/kraken2-db/ .`

## Download the dataset:
`curl -L -o metagenomic-read-files.tar.gz https://ndownloader.figshare.com/files/24079451`

## Unpack the dataset:

tar -xzvf metagenomic-read-files.tar.gz

#Setting up the parameters:
kraken2 --db kraken2-db/ --threads 6 \
        --output sample-1-kraken2-out.txt --report sample-1-kraken2-report.txt \
        --paired sample-1-R1.fq.gz sample-1-R2.fq.gz

## Output error1:

Command 'kraken2' not found, did you mean:

  command 'kraken' from deb kraken

Try: apt install <deb name>

## Output 1 (Results - Bracken):

Loading database information... done.
2 sequences (0.00 Mbp) processed in 0.001s (80.8 Kseq/m, 25.84 Mbp/m).
  2 sequences classified (100.00%)
  >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample-1-kraken2-report.txt -o sample-1-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-05-2020 22:27:28
BRACKEN SUMMARY (Kraken report: sample-1-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 1 
          >> Number of species with reads > threshold: 1 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 2
          >> Total reads kept at species level (reads > threshold): 1
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 1
          >> Reads not distributed (eg. no species above threshold): 0
          >> Unclassified reads: 0
BRACKEN OUTPUT PRODUCED: sample-1-bracken-out.tsv
PROGRAM END TIME: 08-05-2020 22:27:28
  Bracken complete.

## Automation Process
### Making the sample-list.txt file

for sample in $(cat sample-list.txt)
do
    echo $sample
    # what we want the kraken --output file to be
    echo "This is what the regular kraken output file will be named: ${sample}-kraken2-out.txt"
    # what we want the kraken --report file to be
    echo "This is what the kraken report file will be named: ${sample}-kraken2-report.txt"
    # trying to pass the forward read file
    echo "This is where we think the input forward read file is: ${sample}-R1.fq.gz"
    # we can check it exists with the ls command
    ls ${sample}-R1.fq.gz
    # trying to pass the reverse read file
    echo "This is where we think the input reverse read file is: ${sample}-R2.fq.gz"
    # we can check it exists with the ls command
    ls ${sample}-R2.fq.gz
    # and just adding so space between each iteration
    echo ""
    echo ""
done

### Full Automation

for sample in $(cat sample-list.txt)
do
    echo On: $sample
    kraken2 --db kraken2-db/ --threads 6 \
            --output ${sample}-kraken2-out.txt --report ${sample}-kraken2-report.txt \
            --paired metagenomic-read-files/${sample}_R1_trimmed.fastq.gz metagenomic-read-files/${sample}_R2_trimmed.fastq.gz
    bracken -r 150 -d kraken2-db/ -i ${sample}-kraken2-report.txt -o ${sample}-bracken-out.tsv
done
CpuUse=0
MemUse=0
DiskUse=$(du -sh metagenomic-read-files | awk '{print $1}')
Count=0
tempCPU=$(top -H -b -n1 -d1 | grep "Threads" | awk '{print $4}')
tempMem=$(free -g |grep "Mem:" | awk '{print $2}')
CpuUse=`expr $CpuUse + $tempCPU`
if [ $tempMem -gt $MemUse ]
then
        MemUse=$tempMem
fi

Count=`expr $Count + 1`
echo `expr $CpuUse / $Count`
echo $MemUse
echo $DiskUse

### Time Command

command time -f "\t%E real, \t%U user, \t%S sys, \t%M max_mem, \t%K av_mem, \t%P cpu" -o usage-info.txt bash kraken-bracken.sh

## Results

On: sample1
Loading database information... done.
4283096 sequences (1199.29 Mbp) processed in 48.627s (5284.8 Kseq/m, 1479.78 Mbp/m).
  3949426 sequences classified (92.21%)
  333670 sequences unclassified (7.79%)
 >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample1-kraken2-report.txt -o sample1-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-21-2020 20:38:06
BRACKEN SUMMARY (Kraken report: sample1-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 3245 
          >> Number of species with reads > threshold: 3245 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 4283096
          >> Total reads kept at species level (reads > threshold): 2816840
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 1131837
          >> Reads not distributed (eg. no species above threshold): 749
          >> Unclassified reads: 333670
BRACKEN OUTPUT PRODUCED: sample1-bracken-out.tsv
PROGRAM END TIME: 08-21-2020 20:38:06
  Bracken complete.
On: sample2
Loading database information... done.
4327888 sequences (1202.67 Mbp) processed in 51.709s (5021.8 Kseq/m, 1395.50 Mbp/m).
  4049074 sequences classified (93.56%)
  278814 sequences unclassified (6.44%)
 >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample2-kraken2-report.txt -o sample2-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-21-2020 20:46:21
BRACKEN SUMMARY (Kraken report: sample2-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 3092 
          >> Number of species with reads > threshold: 3092 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 4327888
          >> Total reads kept at species level (reads > threshold): 2811244
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 1237198
          >> Reads not distributed (eg. no species above threshold): 632
          >> Unclassified reads: 278814
BRACKEN OUTPUT PRODUCED: sample2-bracken-out.tsv
PROGRAM END TIME: 08-21-2020 20:46:21
  Bracken complete.
On: sample3
Loading database information... done.
4283096 sequences (1199.29 Mbp) processed in 52.500s (4895.0 Kseq/m, 1370.62 Mbp/m).
  3949426 sequences classified (92.21%)
  333670 sequences unclassified (7.79%)
 >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample3-kraken2-report.txt -o sample3-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-21-2020 20:54:10
BRACKEN SUMMARY (Kraken report: sample3-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 3245 
          >> Number of species with reads > threshold: 3245 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 4283096
          >> Total reads kept at species level (reads > threshold): 2816840
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 1131837
          >> Reads not distributed (eg. no species above threshold): 749
          >> Unclassified reads: 333670
BRACKEN OUTPUT PRODUCED: sample3-bracken-out.tsv
PROGRAM END TIME: 08-21-2020 20:54:11
  Bracken complete.
On: sample4
Loading database information... done.
5270348 sequences (1444.14 Mbp) processed in 67.224s (4704.0 Kseq/m, 1288.96 Mbp/m).
  4102225 sequences classified (77.84%)
  1168123 sequences unclassified (22.16%)
 >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample4-kraken2-report.txt -o sample4-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-21-2020 21:02:14
BRACKEN SUMMARY (Kraken report: sample4-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 3588 
          >> Number of species with reads > threshold: 3588 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 5270348
          >> Total reads kept at species level (reads > threshold): 2929872
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 1171564
          >> Reads not distributed (eg. no species above threshold): 789
          >> Unclassified reads: 1168123
BRACKEN OUTPUT PRODUCED: sample4-bracken-out.tsv
PROGRAM END TIME: 08-21-2020 21:02:14
  Bracken complete.
On: sample5
Loading database information... done.
4998731 sequences (1259.68 Mbp) processed in 45.906s (6533.5 Kseq/m, 1646.43 Mbp/m).
  3853084 sequences classified (77.08%)
  1145647 sequences unclassified (22.92%)
 >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample5-kraken2-report.txt -o sample5-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-21-2020 21:08:26
BRACKEN SUMMARY (Kraken report: sample5-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 4623 
          >> Number of species with reads > threshold: 4623 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 4998731
          >> Total reads kept at species level (reads > threshold): 3289608
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 563285
          >> Reads not distributed (eg. no species above threshold): 191
          >> Unclassified reads: 1145647
BRACKEN OUTPUT PRODUCED: sample5-bracken-out.tsv
PROGRAM END TIME: 08-21-2020 21:08:26
  Bracken complete.
On: sample6
Loading database information... done.
4998731 sequences (1249.68 Mbp) processed in 37.057s (8093.5 Kseq/m, 2023.38 Mbp/m).
  3842226 sequences classified (76.86%)
  1156505 sequences unclassified (23.14%)
 >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample6-kraken2-report.txt -o sample6-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-21-2020 21:13:36
BRACKEN SUMMARY (Kraken report: sample6-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 4514 
          >> Number of species with reads > threshold: 4514 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 4998731
          >> Total reads kept at species level (reads > threshold): 3273618
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 568384
          >> Reads not distributed (eg. no species above threshold): 224
          >> Unclassified reads: 1156505
BRACKEN OUTPUT PRODUCED: sample6-bracken-out.tsv
PROGRAM END TIME: 08-21-2020 21:13:37
  Bracken complete.
On: sample7
Loading database information... done.
4998734 sequences (1259.68 Mbp) processed in 43.783s (6850.3 Kseq/m, 1726.27 Mbp/m).
  3813625 sequences classified (76.29%)
  1185109 sequences unclassified (23.71%)
 >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample7-kraken2-report.txt -o sample7-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-21-2020 21:18:22
BRACKEN SUMMARY (Kraken report: sample7-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 4433 
          >> Number of species with reads > threshold: 4433 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 4998734
          >> Total reads kept at species level (reads > threshold): 3347470
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 465937
          >> Reads not distributed (eg. no species above threshold): 218
          >> Unclassified reads: 1185109
BRACKEN OUTPUT PRODUCED: sample7-bracken-out.tsv
PROGRAM END TIME: 08-21-2020 21:18:22
  Bracken complete.
On: sample8
Loading database information... done.
4998734 sequences (1249.68 Mbp) processed in 37.208s (8060.7 Kseq/m, 2015.18 Mbp/m).
  3801365 sequences classified (76.05%)
  1197369 sequences unclassified (23.95%)
 >> Checking for Valid Options...
 >> Running Bracken 
      >> python src/est_abundance.py -i sample8-kraken2-report.txt -o sample8-bracken-out.tsv -k kraken2-db/database150mers.kmer_distrib -l S -t 0
PROGRAM START TIME: 08-21-2020 21:22:35
BRACKEN SUMMARY (Kraken report: sample8-kraken2-report.txt)
    >>> Threshold: 0 
    >>> Number of species in sample: 4328 
          >> Number of species with reads > threshold: 4328 
          >> Number of species with reads < threshold: 0 
    >>> Total reads in sample: 4998734
          >> Total reads kept at species level (reads > threshold): 3331565
          >> Total reads discarded (species reads < threshold): 0
          >> Reads distributed: 469565
          >> Reads not distributed (eg. no species above threshold): 235
          >> Unclassified reads: 1197369
BRACKEN OUTPUT PRODUCED: sample8-bracken-out.tsv
PROGRAM END TIME: 08-21-2020 21:22:36
  Bracken complete.
1
118
5.0G

### Usage Info

22:44.40 real,  1447.33 user,   368.05 sys,     49824956 max_mem,       0 av_mem,       133% cpu