I can't merge my BCF files together using bcftools. Below are the details of my pipeline. After running the pipeline, I created a subdirectory that has 2 *.bcf files to try and merge them as a test set but it's not working.

My commands to merge 2 *.bcf files

 # Directory contents
-bash-4.1$ cd bcf_files/testing/
-bash-4.1$ ls
S-1409-57.B_RD1.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf  S-1410-81.A_RD1.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf   

# Attempting to merge 2 bcf files
-bash-4.1$ bcftools view S-1409-57.B_RD1.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf  S-1410-81.A_RD1.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf > testing.merged.bcf

#Error below
Index required, expected .vcf.gz or .bcf file: S-1409-57.B_RD1.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf
Failed to open or the file not indexed: S-1409-57.B_RD1.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf

I tried indexing them

$ bcftools index S-1409-57.B_RD1.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf
[E::main_vcfindex] bcf_index_build failed for S-1409-57.B_RD1.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf

My pipeline: I have 88 samples whose reads together total to about 746 G in size.

I used HISAT2 for the mapping using human assembly hg38. HISAT2 supplies preindexed files that we used located at ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/grch38.tar.gz

The assembly for the genome used for the indexing was retrieved from ftp://ftp.ensembl.org/pub/release-84/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz

Create the sam file

hisat2 -q -p 2 --fast -x ./grch38/genome -1 {r1_path} -2 {r2_path} -S ./sam_files/{lib}.kneaddata.paired.human.bowtie2.R1-R2.sam

Sam => Sorted-bam

samtools view -bS ./sam_files/{lib}.kneaddata.paired.human.bowtie2.R1-R2.sam | samtools sort -@ 16 -o ./sorted_bam_files/{lib}.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted

Sorted-bam => BCF samtools mpileup -uf ./grch38/Homo_sapiens.GRCh38.dna.primary_assembly.fa -C 50 --BCF -o ./bcf_files/{lib}.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted.bcf ./sorted_bam_files/{lib}.kneaddata.paired.human.bowtie2.R1-R2.sam.bam.sorted

$ du -sh *
5.8T    bcf_files
8.5G    grch38
4.7G    grch38.tar.gz
435K    reads
34K run_tmp.sh
176G    sam_files
38G sorted_bam_files